Covalent organic frameworks (COFs), synthesized from organic monomers, are porous crystalline polymers. Monomers get attached through strong covalent bonds to form 2D and 3D structures. The adjustable pore size, high stability , and metal-free nature of COFs make their applications wider. This review article briefly elaborates the synthesis, types, and applications  of COFs. Furthermore, the applications of COFs as biomaterials are comprehensively discussed. There are several reported COFs having good results in anti-cancer and anti-bacterial treatments. At the end, some newly reported COFs having anti-viral and wound healing properties are also discussed. Crystalline porous materials containing light elements connected reversibly in two to three dimensions through covalent bonds is a developing category of Covalent Organic Frameworks (COFs) . Due to Two-dimensional (2D) COFs;Three-dimensional (3D) COFs.Using different energy resources, COFs can be synthesized. Stable crystalline products which are thermodynamically stable can be synthesized using thermal energy . High en2g−1 Brunauer–Emmett–Teller (BET) surface area greater than the solvothermal synthesized surface area, which is 1590 m2g−1. Similarly, Boronate ester-linked COF (BTD-COF), which is mesoporous, was synthesized by Bein’s group through microwave-assisted synthesis. Under two sequential heatings for 40 min, a BTD-COF with a 1000 m2g−1 BET surface area was synthesized, which is highly crystalline  co-condensation produced 3D COF-202 with butyl functional groups via borosilicate formation . CO. CO98]. Lin’s team used a one-pot approach for assembling COFs and pharmaceuticals in the COF drug-loading system, combining DOX and TAPB-DMTP-COF  99]. DO. DO99]. Protein therapy is a contentious issue in biomedical research. Protein medicine, in contrast to traditional small-molecule treatments, have good targeting, appropriate protection, and substantial efficiency. However, approximately 70% of the genome-encoded proteins faced trouble passing across the cell membrane, resulting in significant challenges for creating protein medicines and discovering new protein activities . COFs’ s–1, which is a low detection limit (LOD). For MCF-7 cells, the aptasensor based on p-COF had a LOD of 61 cell mL–1. The following factors were responsible for the high sensitivity and selectivity . W. WP. aeromposite . Quaternsolution . Qu and reatment ,148.3C2/TpPa-1/Ag composite material with efficient bactericidal properties. The material’s stability was considerably improved by the covalent connection between MXene and COF. Antibacterial activity of Ti3C2/TpPa-1/Ag composite against S. aureus and P. aeruginosa was outstanding [3C2/TpPa-1/Ag demonstrated that electron transfer channels established between the ternary materials might improve carrier separation efficiency and extend the life of photogenerated carriers. Calculations using density functional theory revealed that synergistic catalytic action of Ag and Ti3C2 may significantly decrease the work function along the interface, and a built-in electric field in among layers promotes rapid movement of the carrier, leading to improved catalytic performance [Wang et al. used the Schiff base reaction to covalently connect two materials, MXenes and COFs, and attached Ag nanoparticles (NPs) to create a Tistanding . The fluformance .Staphylococcus aureus and Escherichia coli with a 99% inhibitory efficiency for 50 h. Wound healing therapy effectiveness was investigated using a full-thickness skin defect model in mice. It was indicated that by downregulating inflammatory cytokines (IL-1 and TNF-) and enhancing the expression of growth factors, the ENR–FM–COF–TPU membrane might greatly speed up and promote wound healing (VEGF and EGF). The ENR–FM–COF–TPU membrane offers considerable possibilities in wound healing applications due to its exceptional characteristics [Bacterial infections and inflammations usually accompany skin wounds, resulting in prolonged wound healing, which remains a major therapeutic challenge. As a result, it is essential to produce wound dressings that restrict bacterial infections and speed up wound healing . The proeristics .The condensation process and the Schiff base reaction are used to make a curcumin-loaded COF (CUR@COF), which is then electrospun onto polycaprolactone (PCL) nanofibrous membranes (CUR@COF/PCL NFMs) to create a pH-triggered drug release platform for wound dressing. CUR@COF has 27.68% CUR loading capacity, and CUR@ COF/PCL NFMs have better thermal durability, mechanical characteristics, biocompatibility, and antibacterial and antioxidant activities. More crucially, CUR@COF-based membranes exhibit a pH-responsive CUR release profile through protonation under acidic conditions, implying that an acidic extracellular milieu promotes CUR release from membranes. CUR@COF/PCL NFMs can speed up wound healing and skin regeneration by reducing the expression of inflammatory factors (TNF-a) and increasing the expression of angiogenesis according to histopathological examination and immunofluorescence labeling of an in vivo skin defect model (VEGF) .Escherichia coli and Gram-positive bacteria Staphylococcus aureus in vitro was successfully manufactured. TP-Por CON@BNN6 possesses promising biocompatibility and biodegradability, low phototoxicity, anti-inflammatory capabilities, and outstanding wound healing abilities in mice in vivo. The TP-Por CON@BNN6-integrated heterojunction with multifunctional capabilities appears to be a promising technique for COF-based gaseous therapy and microorganism-infected chronic wound healing [As a result, a new TP-Por CON@BNN6-integrated heterojunction for destroying Gram-negative bacteria  healing .The latest COVID-19 pandemic has sparked global health worries. To combat the COVID-19 pandemic, stopping severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) activity in the body is a promising strategy . One of A molecular dynamics simulation is used to provide an atomic characterization of interconnections between ACE2 and the spike protein in this case . First, COFs have a porous framework and are more versatile than any other porous materials. The development of COFs is still in its early stages. The unique physical and chemical properties make them more reliable to use, especially regarding high chemical and thermal stabilities. Strong covalent bonding is present between the linkers; hence, COFs can be modified easily. Organic linkers such as FGs undergo polymerization to form COFs. COF-derived materials can be used in different applications, including environmental remediation and catalysis.The unique characteristics of COFs are their extremely porous nature and stability. These characteristics make COFs more powerful tools for environmental remediations. Due to their porous nature, the adsorption, filtration, and separation of different components through COFs is made possible. The large porous size allows bulky organic molecules to pass through them. COFs do not contain metal atoms/ions in them, which is another powerful feature of COFs to be used as environmental remediators. COFs are ecofriendly in many ways. For the removal of contaminants in water, COFs are used for degradation. With the help of luminescent COFs, even low concentrations of contaminants can be detected.The use of COFs as biomaterials is still in its early stages, it is essential to determine specific areas where COFs could be useful. COFs, like polymeric biomaterials, can be designed and functionalized in a variety of ways. Medicinal components can be loaded into COFs through built-in and tunable porosity channels. To accomplish functional roles, the drug entrapment amount and release rate of COFs can be modified within a specific range by systematically adjusting pore size and polarity. COFs can be employed to enhance the photothermal effect of nanomaterials or as transporters for photothermal agents in photothermal therapy. In contrast to a closed porous structure, which impedes heat transfer, COFs can rapidly transport temperature. COFs possess high load capacity because of their systematic pore structure and high specific surface area. COFs are employed to transport drugs to the target in drug chemotherapy. COFs with a wide surface area and porosity, high electrical conductivity, and novel interactions with bioactive compounds could diagnose (biosensing and bioimaging) cancer.2 reduction, water splitting, radical formation, and organic transformation reactions. COF-based catalysts possess better structural activity relationship understanding, good confinement effect, size sieving and mass transfer, and better access of substrate concentration and catalytic site due to their high surface area. With COF-based catalysts, catalytic reactions can proceed through different mediums. Catalytic sites can be easily installed, and guest species can be incorporated easily due to manageable pore space.COFs can be used for metal catalytic applications  as they do not contain metal atoms/ions in them. COFs have high thermal and chemical stabilities with adjustable active sites. These properties make COFs ideal porous catalysts. COFs such as COFs with reactive metals, COFs with molecular catalysts, COFs with reactive linkages, and COFs with reactive pendant groups can be made either in post-synthetic, in situ or bottom-up methods. COFs have been used for various catalytic processes like photocatalysis, chemical catalysis, and electrocatalysis for almost a decade. Two-dimensional and three-dimensional COF- based catalysts with unique structural and chemical features have been designed to catalyze different reactions, such as redox reactions, organic coupling reactions, degradation, and addition reactions. COFs showed better catalytic activities than other catalysts. COF base photocatalysts have been designed with photoactive linkages by installing photoactive metals in the porous confinement of COF. The photocatalytic COFs are used for organic redox reactions, CODespite all the efforts made for the development of COFs, the development is still in its early stages. There is still more to be developed; for example, 3D COFs in comparison to 2D variants are less explored. The development of 3D COFs is extensively needed for photocatalytic and electrocatalytic applications.The following challenges must be addressed: Facile methods should be explored for COF synthesis. The assembly of COF-based functional materials should be investigated, and a relationship should be established between structure and performance. Cost effective linkers should be selected so that further advancement of COFs can be done. COFs should be assessed against environment hazards. Recycling and reuse of COF-based materials should be addressed for more advancements in COFs.


C. elegans, which does not have a complement system, suggesting an ancient function. Comparison of the roles of CUB-, CCP-, and TSP-1 containing synaptic proteins in various species shows that in more complex nervous systems, these structural domains are combined with other domains and that there is partial conservation of their function. These three domains are thus basic building blocks of the synaptic architecture. Further studies of structural domains characteristic of synaptic proteins in invertebrates such as C. elegans and comparison of their role in mammals will help identify other conserved synaptic molecular building blocks. Furthermore, this type of functional comparison across species will also identify structural domains added during evolution in correlation with increased complexity, shedding light on mechanisms underlying cognition and brain diseases.The appearance of synapses was a crucial step in the creation of the variety of nervous systems that are found in the animal kingdom. With increased complexity of the organisms came a greater number of synaptic proteins. In this review we describe synaptic proteins that contain the structural domains CUB, CCP, or TSP-1. These domains are found in invertebrates and vertebrates, and CUB and CCP domains were initially described in proteins belonging to the complement system of innate immunity. Interestingly, they are found in synapses of the nematode  Caenorhabiditis elegans by Sydney Brenner identified many genes required for synaptic function, such as unc-13, which encodes a protein essential for synaptic vesicle exocytosis . The seminal work of the laboratory of Dr. Carla Shatz, followed by others, showed that proteins of the immune system are also expressed by neurons, are regulated by activity and contribute to synapse development and function (for a review cf. ). One paIn line with this idea, Vogel and Clothia studied 38 eukaryotic genomes, ranging from unicellular organisms to mammals, and tested the correlation between the number of proteins in superfamilies characterized by specific structural domains and the complexity of the organisms as defined by the number of cell types . They idCaenorhabditis elegans were instrumental for the identification of new synaptic proteins and their function at the neuromuscular junction (NMJ). In particular, screens using the anthelmintic drug levamisole identified two genes, lev-9 and lev-10, coding for synaptic proteins required for the aggregation of levamisole-sensitive cholinergic receptors (L-AChRs) on muscle cells (madd-4 was identified as a gene encoding a protein composed of up to 10 thrombospondin type-1 (TSP-1) domains  was first described in thrombospondins, multimeric glycoproteins present at the cell surface and in the extracellular matrix. It is composed of an antiparallel, three-stranded fold with a positively charged groove . The CCPequences . The CUBequences . The conequences  often coequences .All three domains were present in the last common ancestor of eumetazoans, as shown by their presence in bilaterians (described in this review) and in cnidarians (SMART website). Proteins containing these structural domains can thus be expected to play ancient and key roles in the building and function of neuronal circuits.C. elegans, the NMJ has been used as a model to study synapse specificity on body-wall muscle cells, which receive both excitatory (ACh) and inhibitory (GABAergic) inputs. MADD-4, a TSP-1 domain containing protein, is secreted presynaptically by motoneurons and defines the identity of postsynaptic domains by controlling the clustering of cholinergic receptors (AChRs) and GABAergic A receptors (GABAARs) in front of the corresponding neurotransmitter release sites  differ by 2 amino acids and are composed of 10 TSP-1 repeats, an ADAMTS  cysteine-rich module, an ADAMTS spacer module, an Ig-like C2-type domain and a PLAC (Protease and LACunin) domain  domain . The MAD) domain . MADD-4Lic areas . Hence, ic areas . Recentlelopment .ARs onto NLG-1 clusters. In addition, the mechanism allowing the dual function of MADD-4S at cholinergic versus GABAergic synapses remains to be understood, but probably involves dimerization with the long isoform MADD-4L. Finally, both MADD-4 isoforms promote the synaptic localization of the heparan sulfate glycoprotein syndecan/SDN-1, a key-component of cholinergic NMJs  in front of cholinergic boutons . MADD-4Sgic NMJs . The spelev-9 mutants, while ACh presynaptic boutons and postsynaptic GABAARs are normally distributed  domain . L-AChRstributed . Surpris L-AChRs . However mutants . These d mutants . LEV-9 a mutants . The WAP mutants , indicatDrosophila melanogaster: Hikaru genki (Hig) and Hasp (Hig-anchoring scaffold protein). The Hig protein contains five CCP domains and one immunoglobulin (Ig)-like domain, while Hasp contains one WAP domain followed by up to seventeen CCP domains  homolog (STG-1 or STG-2) and SOL-1 . For example, the number of proteins containing at least one CCP domain in C. elegans is 11, representing 0.14% of the total pool of proteins, while in Rattus norvegicus, 54 CCP-containing proteins are found, representing 0.71% of the total pool of proteins. This suggests a correlation between the number of proteins containing these domains and organism complexity. This relationship is true for the CUB domain when numbers are compared with D. melanogaster, but not with C. elegans where a large number of CUB domain-containing proteins are found, suggesting a specific evolution in this organism , which are large secreted extracellular matrix (ECM) proteins that mediate cell–cell and cell–ECM interactions (reviewed in ). Amongsd by P21 . Both TSd by P21 . In the d by P21 .via its TSP-1 domains (via its TSP-1 domain.TSPs have many interacting partners (reviewed in ), includ domains . Beta-in domains . TSP1 al domains . Further domains , indepenThe three brain-specific angiogenesis inhibitor receptors BAI1-3,  are a suin vivo  was identified as the causal gene for Rolandic  epilepsy  and encoP domain , showingSrpx2 is expressed during the development of the cortex , suggesting a role for these other SUSD members in brain development and/or function.The Sushi domain-containing protein (SUSD) family is composed of six proteins, which all contain one or more CCP domains , and, exsynapses  and loca numbers . Loss-ofairments  and misrairments . SUSD4 iubfamily . These uubfamily . Loss-ofnduction . Thus innduction . Interesnduction  : given Bcan knockout mice are dramatically impaired for the maintenance of synaptic long-term potentiation (LTP), while the basic properties of excitatory and inhibitory synapses as well as LTP induction are normal  and cone normal . In the y normal . Experimy normal . CCP domDrosophila Neto). They are both type 1 transmembrane proteins with a large ectodomain composed of one LDLa domain and two CUB domains (+-Cl– cotransporter KCC2 (NETO1 and NETO2 are brain-specific proteins with a domain organization resembling the one of CUB-domain containing proteins in invertebrates (LEV-10, SOL-2 and  domains , 2004. T domains . The CUB domains , kainate domains  and the ter KCC2 . NETO2 iter KCC2  while NEter KCC2 . Both NEter KCC2 .Neto1–/– mice but not Neto2–/– mice (Neto1–/– mice present NMDAR defects at specific synapses: NMDAR mediated currents are reduced at CA1/Schaffer collateral synapses and at associational/commissural CA3 synapses but not at mossy fiber CA3 synapses (Neto1 or Neto2 (In vertebrates, NETO1 and NETO2 have been primarily studied for their role as auxiliary subunits of kainate receptors . Coexpre–/– mice . Moreove–/– mice . In addisynapses . No defeor Neto2 .Neto1 knockout mice, GluN2A levels are reduced in PSD fractions but not in whole brain extract, and surface GluN2A are unchanged (Neto1 leads to loss of presynaptic kainate receptors at immature CA3-CA1 synapses and a deficit in synaptogenesis (Besides their role in regulating glutamate receptor function, NETO proteins could also regulate receptor localization, although the evidence for this role is contradictory. While some studies did not detect any changes in KAR abundance at synapses in the absence of NETO proteins , others nchanged . This suogenesis . Thus, bNrp2 in mice leads to increased epileptogenicity (Nrp-2 and Sema3F, a ligand of NRP2 (Nrp-2 knockout mice, patch-clamp recordings found increased mEPSC frequency in cortical and hippocampal neurons in the absence of changes in the paired-pulse ratio (The Neuropilin family is composed of two members, neuropilin-1 and neuropilin-2 (NRP-1 and NRP-2, respectively, genicity . Increas of NRP2 . In the se ratio  and decrse ratio , indicatse ratio . NRP-2 ise ratio . These rC. elegans suggest a role for TSP-1 containing proteins in synapse specification on a given target cell (C. elegans, suggesting that the appearance of this structural domain might have contributed to the increased diversity of synapse types in vertebrates. Interestingly, while CCP, CUB, and C1Q domain containing proteins were identified first in the immune system, the presence of CCP and CUB domain proteins in C. elegans suggest an ancient role for these structural domains in forming and regulating signaling complexes, in particular with neurotransmitter receptors, a role that could have been then tethered to various biological systems during evolution.Comparing the structure of CCP, CUB, and TSP-1-containing proteins across species shows that invertebrates contain synaptic proteins with stretches of a single domain type whereas mammalian synaptic proteins are composed of combinations of CCP, CUB, and TSP-1 domains, together with a large diversity of other domains . This suget cell  and the get cell . C1QL1 iget cell . The C1qCCP and CUB domains are combined in neuronal proteins such as SEZ6 isoforms  and CSMDHomo sapiens, the SUSD4 gene is located in the chromosome deletion linked with Fryns syndrome, which is an autosomal recessive multiple congenital neurodevelopmental disorder associated with intellectual disability (SUSD4 variants have also been associated with autism spectrum disorders (ADGRB1-3 genes have been associated with disorders such as autism spectrum disorders (ADAMTSL3, the human orthologue of C. elegans MADD-4, and all three CSMD genes have been associated with schizophrenia (The percentage of proteins containing CCP, CUB, and TSP-1 domains is increased in vertebrates compared to invertebrates . Many fasability  and SUSDisorders . ADGRB1-isorders , schizopisorders , bipolarisorders , intelleisorders  and addiisorders . Variantophrenia . UnderstIG-C, MC, J-LB, and FS contributed to conception of the manuscript. IG-C, MC, and FS wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt’s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds . Here, we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver. The Ser/Thr protein kinase Akt1 (termed Akt in the present work) and its paralogs Akt2 and Akt3 serve as key effectors in the PI3-kinase/Akt signaling pathway to regulate cell survival, proliferation, and metabolism by phosphorylating a variety of protein substrates . Akt is In the current structural model, even for an isolated Akt kinase domain, phosphorylation of Thr308 is critical to reach its active catalytic conformation whereas pS473 effects are only apparent in the full-length enzyme and relieve inhibitory intramolecular interactions between the kinase and PH domain . The pS4PIP3 production is governed by the action of receptor tyrosine kinases like the insulin receptor which in the presence of agonists stimulate PI3-kinase that catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to PIP3 . PIP3 hyRecent efforts to interrogate Akt’s molecular mechanisms have relied on the technique of expressed protein ligation (EPL) to generate site-specific C-tail modified forms of Akt . This meSeveral prior studies have attempted to elucidate the structural basis of PH domain-mediated autoinhibition of Akt, and two types of high-resolution structures of near full-length Akt in distinct ‘closed’ conformations have been obtained by X-ray crystallography . These XIn this study, we have explored the PH domain-mediated autoinhibition of Akt using an array of biochemical and structural approaches. Key to this work was the unexpected identification of an Akt PH domain mutant, R86A, which we posit sheds light not only on PH domain-mediated Akt autoinhibition but also on how the oncogenic mutant E17K activates Akt.–1) and R15A/K20A/R67A Akt  forms showed similar kinase rates to WT Akt  prepared under the same conditions (–1) displayed an approximately fourfold reduction in enzymatic activity. This suggested that the Arg86 side chain would be a candidate for electrostatic interaction with pS477 and/or pT479 to induce Akt activation. To explore this further, we generated R86A pS473 and non-phosphorylated C-tail Akt and unexpectedly found that Arg86 mutation suppressed the catalytic activity of pS473. In this set of measurements R86A pS473  was about fourfold lower than WT pS473   . These rD 37 μM) toward the kinase domain than the WT PH domain (KD 107 μM), supporting the idea that R86A mutation stabilizes the autoinhibited Akt state by promoting the PH-kinase interdomain interaction. We also attempted to measure the affinity of the R86A PH domain to the kinase domain in the presence of the Akt allosteric inhibitor MK2206 using MST but were unable to see detectable binding (KD > 250 μM). This was an unexpected result since MK2206 has been shown to enhance the affinity of WT PH domain to the kinase domain, previously  for the Cy5-labeled kinase domain  using microscale thermophoresis (MST). As shown in eviously . We thus15N-labeled PH domain. Remarkably, the 2D 1H-15N HSQC NMR analysis of the R86A PH domain displayed significant chemical shift perturbations (CSPs) relative to the WT PH domain  for the R86A mutant  full-length R86A Akt using our semisynthetic strategy. Here, 15N-labeled R86A PH domain was ligated to the Akt kinase domain harboring a pS473 in the C-terminal tail. We compared the CSPs on the R86A PH domain due to the interaction of the kinase domain and C-terminal tail with the CSPs on the WT PH domain under the same conditions  and the Y18A/R86A PH domain (KD 210 μM), approximately threefold and approximately sixfold weaker than that of the R86A PH domain (KD 37 μM), respectively (D 45 μM) was very similar to that of the R86A PH domain (KD 37 μM) and the E17K/Y18F/R86A PH (KD 21 μM) was approximately twofold tighter  and Y18A  were 2.5-fold and 18-fold elevated with respect to WT  whereas the catalytic efficiency of the R86A mutant  was reduced by twofold (cat/KM 0.12 min–1 μM–1) and Y18A  were 1.7-fold and 15-fold increased compared to WT  which was threefold greater than R86A . These results are consistent with the dominance of Tyr18 in mediating Akt autoinhibition, even when partially relieved by pS473  was essentially identical to WT (KD 0.26 μM) and much weaker for Y18A (KD 4.5 μM) and R86A , similar to WT (D 5.6 μM) was approximately fivefold tighter relative to its engagement of the WT PH domain  PIP3 using fluorescence anisotropy. It was found that PIP3 affinity for E17K (Kar to WT . These r1/2 60 min–1) and Y18A  Akt forms displayed about 2.5-fold slower dephosphorylation, respectively, relative to WT Akt  (1/2 19 min–1) was slightly (but not significantly) faster than WT Akt  . In contn WT Akt . Alkalinn WT Akt . Taken tAs described above, we have obtained strong evidence that Tyr18 is a critical player in the autoinhibitory interaction between PH and kinase domains. Inspection of prior X-ray crystal structures of near full-length Akt in complex with allosteric inhibitors shows that Tyr18 is in close proximity to the catalytic core of the Akt kinase domain , but we –1) was found to be approximately twofold greater than WT Akt  was demonstrated to be 40% lower compared with WT PH-free Akt  mutants that constitutively localize to the plasma membrane regardless of PH-mediated PIP3 binding status . This moThe cellular activity of Akt was determined by the phosphorylation level of Akt substrate Foxo1/3a (p-Foxo1/3a) using western blot analysis. As expected, non-Myr WT Akt showed just comparable p-Foxo1/3a level with that of non-transfected control, and the incorporation of myristoylation signal sequence on WT Akt was effective in increasing its phosphorylation level at pT308 . DespiteCapturing a detailed picture of the autoinhibited state of Akt has remained a challenge for over two decades . As discIntending to characterize how the non-canonical pS477/pT479 activates Akt, we stumbled on a critical hydrogen bond within the PH domain that govern its stability and affinity for the kinase domain. Paradoxically, breaking the Arg86-Glu17 hydrogen bond by R86A mutation sharply increases PH domain stability and promotes a Tyr18 rotamer which enhances the binding of the PH domain to the kinase domain. It is the phenyl ring rather than the hydroxyl that mediates its interactions with the kinase domain. Although our studies do not provide definitive information on how pS477/pT479 activates Akt, it is still possible that these phosphorylations may do so by directly engaging Arg86.Our investigation of the role of Arg86 has also led to a deeper understanding of the molecular mechanisms associated with the well-known Akt E17K oncogenic mutant. At first glance, it appears paradoxical that replacing Glu17 with Lys would be activating, since, like R86A, this mutation disrupts the Glu17-Arg86 salt bridge, and as discussed R86A promotes autoinhibition. We believe the key difference relates to the structural properties of Lys17. In a previously determined X-ray crystal structure of the E17K Akt PH domain, the Lys17 side chain makes a hydrogen bond with the Tyr18 phenol and restrains the Tyr side chain from engaging the Akt kinase domain . Thus, iA combination of enzymology, mutagenesis, AlphaFold, and fluorine NMR experiments point to an unexpected model in which Tyr18 participates in a stabilizing interaction with the side chain of Phe309. The AlphaFold-predicted structure appeared unlikely initially since the activation loop containing Phe309 is commonly considered quite flexible and the prediction for the PH-kinase interdomain interface is limited . HoweverOur approach to generating specific, chemically modified Akt forms has involved EPL to generate site-specific C-tail modified forms of Akt. Although it has been suggested by Truebestein et al. that EPL may result in unstable forms of Akt due to a lack of Thr450 phosphorylation , our recIn summary, through a combination of structural and biochemical approaches, we have developed a new model for how Akt is autoinhibited by its PH domain and how E17K relieves such autoinhibition. It is hoped that the mechanistic insights here may pave the way toward novel targeted inhibitors that can exploit the distinct features of E17K Akt that can be used for the treatment of cancer.Mxeintein-CBD (CBD: chitin binding domain) constructs (Akt1 aa 1–459 and 122–459) according to the previously reported procedures , the cells were lysed by french press and the resulting lysate was cleared by centrifugation at 15,000× g for 40 min at 4°C. The insect cells expressing Akt -MxeIntein-CBD were resuspended in lysis buffer and lysed in a 40 mL Dounce homogenizer on ice, and the resulting lysate was cleared by centrifugation at 15,000× g for 40 min at 4°C. Next, both N-Tags-TEV-S122C-Akt kinase domain (aa 122–459)-MxeIntein-CBD  and N-terminally His-tagged Akt PH domain (aa 1–121)-MxeIntein-CBD proteins were purified by chitin bead-mediated affinity chromatography from the cell lysates. The protein C-terminal thioester forms of both the Akt PH and kinase domains were eluted via intein cleavage using MESNA (sodium mercaptoethylsulfonate) according to established protocols  having variable phosphorylation in the first ligation buffer  for 5 hr at room temperature and then maintained overnight at 4°C. The preparation of the recombinant GST-PDK1 and the synthetic C-terminal Akt peptides were described in our previous work . The lig15N-labeled Akt PH domain, the Akt (aa 1–121)-MxeIntein-CBD was expressed in E. coli Rosetta (DE3)/pLysS (Novagen) following the established protocol , 100 mg/L ampicillin and 20 mg/L chloramphenicol) in H2O, and was further supplemented with trace elements  and the vitamins biotin (0.5 mg/L) and thiamin (0.5 mg/L) in shaker flasks at 37°C until OD600=0.5. After that, IPTG was added to make a final concentration of 0.5 mM to induce the expression and the culture was further incubated for 18 hr at 16°C. The cells were pelleted and stored in –80°C freezer for the next steps. To incorporate fluoro-Tyr into the Akt PH domain, the Akt -MxeIntein-CBD was expressed in E. coli Rosetta (DE3)/pLysS (Novagen) as previously described  in H2O, and was further supplemented with trace elements  and the vitamins biotin (0.5 mg/L) and thiamin (0.5 mg/L) in shaker flasks at 37°C until OD600=0.5. After that, 1 g of glyphosate, as well as 50 mg of phenylalanine, tryptophan, and 3-fluoro-tyrosine were added. When OD600 = 0.7, 1 mL of 0.5 M IPTG was added to induce the expression, and the culture was further incubated for 18 hr at 16°C. The cells were pelleted and stored in –80°C freezer for the next steps. The three-piece EPL strategy described above was employed to produce full-length Akt containing labeled PH domain (15N or fluoro-Tyr18) for NMR studies.To obtain the protocol . Brieflyescribed . Briefly1H, 13C, 15N TCI cryoprobe, and z-shielded gradients. All data were processed using Topspin (Bruker) or NmrPipe  were accumulated, leading to measurement times of 54 hr. Chemical shifts were calibrated using internal water calibration in NmrPipe. Combined CSPs and associated standard deviations were calculated according to the protocol from If not stated otherwise, NMR experiments were conducted on a Bruker spectrometer operating at 750 MHz, equipped with a triple-channel  NmrPipe  and anal NmrPipe . For NMRficantly . Eighty-ficantly . Chemica1, R2, Etaxy) were measured on a Bruker 800 MHz using standard pulse sequences and recycle delays of 5 s. R2 relaxation employed Carr-Purcell-Meiboom-Gill (CPMG) pulse trains with varied delays and corresponding temperature compensation blocks. All experiments were acquired using single scan interleaving. R2 relaxation delays were set to 10, 30, 50, 70, 90, 130, 150, 170 ms (50 ms was repeated twice). R1 relaxation delays were set to 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 1, 1.2, 1.5, 1.8 s (0.5 s was repeated twice). Cross-correlated (DD/CSA) relaxation rates were measured at a relaxation delay of 50 ms. For steady-state hetNOE, 1H saturation was achieved with a train of 180° pulses at 12 kHz power for a delay of 6 s (>4⋅T1max), recycle delays of 10 s were employed. CPMG relaxation dispersion experiments were conducted with a T2 relaxation delay of 40 ms. CPMG frequencies were modulated in the following order (in Hz): 0, 1000, 50, 850, 100, 650, 500, 250, 400, 100, 50, 850, 400, 1000. Dispersion curves were analyzed with the program Relax.Nuclear spin relaxation , and 64k scans for a total experimental time of approximately 10 hr.MxeIntein-CBD sequence was transformed into E. coli Rosetta (DE3)/pLysS (Novagen) to express corresponding protein construct using the established protocol , and then lysed by french press and the lysate was cleared by centrifugation at 15,000× g for 40 min at 4°C. Next, the Akt PH domain -MxeIntein-CBD protein was purified by affinity chromatography from the cell lysate using chitin beads. After loading onto chitin beads, the C-terminal thioester form of Akt R86A PH domain was eluted via intein cleavage using 30 mM DTT as previously described -protocol . Brieflyescribed , and theThe R86A PH domain protein was concentrated to 10 mg/mL and crystals were grown using the sitting drop method in a 96-well plate by adding a 1:1 volume of well solution  acetate) to protein and incubated at 20°C for 3 days. Crystals were cryoprotected in the same precipitant buffers where they were grown with 20% (v/v) ethylene glycol added, flash-frozen, and stored in liquid nitrogen until data collection.Diffraction data of R86A PH domain protein were collected at APS NE-CAT 24E on a DECTRIS Eiger X 16 M detector. The data were indexed, integrated, and scaled using XSCALE. The R86A PH domain crystals were isomorphous with those of the previously reported WT structure (PDB: 1UNP), which was used as an initial model for refinement . The modWT and R86A PH domains were expressed and purified as previously described. Reactions were prepared in 384-well plates (Greiner Bio-One) in a total volume of 33 μL. SyproOrange dye dilutions in assay buffer  were first prepared by a NT8 liquid handler (Formulatrix) followed by addition of 10 μM PH domain. Dilution series were ×0.2–30 SyproOrange dye . Sealed plates were heated at 1°C/min from 25°C to 95°C with fluorescence reading (586 nm) every 0.5°C in a ViiA-7 real-time PCR system . Tm values were determined as the minimum of the first derivative of the recorded fluorescence intensity versus temperature. Fluorescence intensity at 25°C and melting temperatures were found independent from dye concentration.g for 5 min. Supernatant of each sample was transferred to a low binding liquid chromatography vial, and 100 ng of protein was injected into a Vanquish Flex LC interfaced to a Q Exactive mass spectrometer (Thermo Fisher Scientific). Proteins were separated, ionized, and detected as previously described (Akt proteins in Akt storage buffer were diluted to 50 ng/μL in 50 mM ammonium bicarbonate (pH = 8.0) and centrifuged at 15,000× escribed . For eacescribed .32P-labeled ATP and N-ε-biotin-lysine GSK3 peptide (RSGRARTSSFAEPGGK) as substrates, as previously described with minor modification  in all assays. The kinase assay buffer is composed of 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 1 mM sodium orthovanadate, 0.5 mg/mL BSA, approximately 0.5 µCi γ-32P-ATP, and varying amounts of ATP (0–2.5 mM). The kinase reactions were performed at 30°C for 10 min and quenched by the addition of 100 mM EDTA, and then 100 µg of Avidin (Pierce) was added to each sample and incubated for 20 min at room temperature. Each sample was transferred to a centrifugal 10 kDa MWCO filtration unit  and washed five times with 120 μL of washing buffer . Each filtration unit containing the biotinylated GSK3 peptide substrate complexed with Avidin was placed in 10 mL scintillation fluid and radioactivity came from the 32P-labeled phosphate group on the peptide substrate was measured by Beckman liquid scintillation counter (Beckman LS6500) to determine what percentage of the peptide substrate in each sample was phosphorylated in 10 min. Kinetic parameters of each Akt mutant were determined from V/[E] versus [ATP] plots using the Michaelis-Menten equation. As we have noted previously, some of the moderately different kinase activities here versus prior reports may reflect different levels of linker phosphorylations that can vary among insect cell preparations and in vitro versus in cellulo PDK1 phosphorylation used to generate pT308  was N-terminally Cy5 labeled by pretreating Sulfo-Cy5-NHS ester (Lumiprobe) with MESNA to efficiently convert NHS ester into thioester that can react with the N-terminal cysteine selectively, as previously described . The binD value for each Akt PH domain mutant was obtained by fitting the data to the quadratic binding equation as described before (The binding affinity of the Akt PH domain with phospholipid PIP2 or PIP3 was determined using fluorescence anisotropy. Fifty nM fluorescein-labeled soluble (C8) PIP2 or PIP3  was mixed with varying amounts of Akt PH domain in binding buffer  and the resulting mixture was incubated at room temperature for 30 min. Fluorescent anisotropy spectra were recorded by Multi-Mode Microplate Reader (Biotek Instruments) at 23°C with three different replicates. The Kd before .2) and incubated at 30°C. Ten μL of the reaction was collected at the indicated time points and quenched by the addition of ×2 SDS-loading buffer. The SDS samples were loaded on SDS-PAGE gel for western blot analysis with anti-pT308  or anti-pS473 (Abcam) and pan-Akt  primary antibodies.The dephosphorylation assays were carried out following the protocol previously described with minor modification . BrieflyAfter transferring proteins from gels to nitrocellulose membranes using an iBlot (Thermo Fisher Scientific) system, 5% (w/v) BSA in TBS-T buffer was used to block the membranes for 30 min incubation at room temperature. After that, the membranes were incubated with primary antibodies  at a 1:1000 dilution with 5% (w/v) BSA in TBS-T buffer overnight at 4°C, followed by a 10 min wash three times with TBS-T buffer, and then incubated with secondary HRP-linked secondary antibody  for 1 hr at room temperature, followed by 10 min wash three times. Lastly, the membranes were developed with Clarity Western ECL Substrate (Bio-Rad) and imaged by a GeneSys  imaging system.2. When the cell confluence reached around 70% in six-well plates, the medium was changed with McCoy’s 5A (Gibco) having 5% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco), and the cells were transfected with 1.5 μg of pcDNA3.1-Flag-HA-Akt plasmid or 1.5 μg (3.0 μg for Y18A mutant) of pcDNA3.1-Myr-HA-Akt plasmid complexed with 3 μL Lipofectamine 3000 (Invitrogen) and 3 μL P3000 reagent (Invitrogen) in Opti-MEM medium (Gibco) at 37°C and 5% CO2. Seventy-two hours after transfection, the cells were washed with cold PBS and lysed by adding 150 μL RIPA buffer  containing ×1 complete protease inhibitor tablet (Thermo Fisher Scientific) and ×1 PhosStop tablet (Roche), and 1 mM PMSF for 10 min at 4°C. Thirty μg of total protein (BCA assay) was loaded on an SDS-PAGE gel. Membrane transfer and western blotting were carried out as described above with 1:1000 dilution for primary antibodies: anti-Akt, anti-pT308 Akt, anti-Foxo1, anti-Foxo3a, and anti-pT24 Foxo1/pT32 Foxo3a, anti-GAPDH .The Akt1/2 knockout HCT116 colon cancer cell line was given as a gift from Dr Bert Vogelstein  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Amy Andreotti as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Volker Dötsch as the Senior Editor.Thank you for submitting your article "PH Domain-Mediated Autoinhibition and Oncogenic Activation of Akt" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:There are a number of reviewer suggestions to further strengthen this manuscript (see specific reviewer comments below). In particular, two important issues are identified that the reviewers request be addressed in a revised manuscript:1) Questions are raised about the validity and predictive value of the AlphaFold model. Please see specific reviewer comments below and address them accordingly.2) Further validation of protein samples is requested. Specifically, MS quantification and gel filtration profiles are requested.In addition, the framing of this work creates some confusion. Given that this is submitted as a Research Advance, it is framed in the context of different phosphorylations of the C-tail on AKT. Since the results then focus on side chains in the PH domain the framing, as presented, creates some confusion about the goals at the outset and the eventual findings. The reviewers are confident that the authors can reframe the work in a manner that both explains the connection to earlier findings and clearly steers the reader through the present work.Reviewer #1 (Recommendations for the authors):The following are provided to improve the quality of this manuscript:1) Figure 3A seems to show multiple peaks for the wt protein possibly indicative of slow exchange (the mutant spectrum appears greatly simplified). The authors do not adequately comment on this or attempt to establish whether the resonances that appear to give rise to multiple peaks are structurally related. Moreover, it is unclear whether the wt sample could be subject to changes in temperature or ZZ-exchange experiments to more carefully analyze the possibility of multiple species in slow exchange. This seems relevant to the idea that the R86A mutation 'stabilizes' the PH domain and in so doing pre-organizes the PH domain for binding to the kinase domain.2) The notion that the R86A mutation stabilizes the PH domain seems to be based on the longevity of the protein sample. Could a more direct measure of protein stability that does not require the long acquisition times of the NMR relaxation experiments  be used to support this idea?3) In describing the data for Figure 3—figure supplement 3, the authors state that "we observe similar patterns of CSPs and peak broadening indicative of the interaction of the PH domain with the rest of the protein. Though the comparative CSP patterns of the R86A and WT PH domains when anchored to the rest of Akt look similar, there are subtle differences…". I agree with this conclusion but due to the absence of many peaks in the wt spectrum it is hard to pick out the similarities between the WT and R86A mutant as presented – in fact the histograms in Figure 3—figure supplement 3 look quite different. Perhaps the language used to compare the two datasets can be modified to make the point clearly without creating confusion. Importantly, it seems the authors need to clearly show the differences around residues 67-73.4) With regard to residues 67-73 it is intriguing that this is where the CSPs seem to differ between wt and R86A. Inspection of the structure shows that R69 in this region is involved in very close interactions with W22 (cation π interaction?). As well, R15 sits on the opposite face of W22 and both R15 and W22 surround the important E17-Y18 segment. Moreover, R69 is surrounded by prolines – could proline isomerization be at play here? Certainly, this is an event that could induce the slow exchange that is suggested in the 1H-15N HSQC spectrum of wt .5) In Figure 7A-D.  "PH domain" in blue is the R86A mutant? If so please clarify in the figure and caption – the text indicates the R86A PH domain while the figure is unclear. Assuming Figure 7 is the R86A mutant, was the corresponding data for the wt PH domain also acquired to assess conformational heterogeneity at the Y18 position for wt versus R86A mutant? Such an experiment could help better understand the effects of the R86A mutation on the Y18 sidechain.Reviewer #2 (Recommendations for the authors):The multi-pronged approach systematically investigates the interaction between PH:Kinase, stability/structure of PH domain, activity of AKT and half-life of activation loop phosphorylation. Methodologically this work is a tour de force with the use of 3-way ligation to generate specific, stoichiometric post-translationally modified variants to study and highlights the limitations of studies using non-physiological variants.eLife audience. The main conclusion of the paper comes quite late and was unanticipated. While such findings are exciting, I found it hard to connect the earlier and later results into a coherent finding.This paper is written for an AKT expert and may not successfully connect with the broader eLife audience. For example, an introduction to the regions of the PH domain responsible for lipid binding and those proposed to bind AKT would help provide context for understanding the early mutagenesis. Perhaps figure 1B could be relabeled to identify these surfaces of the PH domain? Similarly, introducing the models of AKT autoinhibition earlier would help provide a framework for the subsequent experiments.– This paper is written for an expert and as such may not successfully connect with the broader – The paper is framed as a study in the role of different phosphorylations of the C-tail on AKT but ends with a result that is separate from the tail. At the end of the paper, I'm still pondering how the tail could/would influence this new autoinhibitory interaction. Can the authors clarify how the phosphorylation of the AKT tail influences interactions/activities of variants tested and how those states would influence their model? Relatedly, several times throughout the paper I struggled to determine whether the variant used had the tail present and, if so, its phospho-state. I'm still not sure if the experiments in Figure 2C/Figure 4A were performed with or without the tail as there is a disconnect between the carton and description. Also, the description of the variants I think varies-- is "non-C-tail phosphorylated Akt" the same as" full-length AKT mutations in the absence of C-tail modification"?– Earlier work from this lab suggests that the linker between the PH and kinase domain influences inhibition. As this region was missing in experiments using the PH domain in trans, could the authors speculate on how the lack of the linker would influence those results?eLife says this loop is important for binding C-tail and PIP3 – what significance does the additional disorder mean? This disordered loop also implies the statement that the two structures of PH domains are in "high concordance" and "identical" is an overstatement (pg7).– Arguments that the PH domain of R86A retains the same fold as WT but is more stable/less dynamic are indirectly and weakly supported. While this claim is not critical to the later arguments, the data presented are not compelling. The authors claim that the WT PH domain was less stable b/c it degraded during the NMR experiments but do not show data supporting that claim. They then conclude the structures are only slightly changed between WT and R86A based on the ~60% of residues they can assign. If the WT PH domain is degrading during the course of the experiment, how legitimate are the assignments from this experiment and how do they know they are looking at a degraded protein? The analysis of the B-factor of the crystal structure also does not provide much insight into dynamics, especially without any comparison to the b-factor distribution of the WT PH domain – A quick look in pymol reveals a similar pattern of B-facto distributions for the WT PH Domain. Further in 7MYX the loop 42-49 is disordered but is well resolved in the WT structure (1UNP). Since Chu 2020 – Can the authors speculate at the precise step that the PH domain is inhibiting AKT? The Α Fold 2 structure looks to have the activation loop in the out (ie activated state) even though there is no phosphorylation on the activation loop for AlphaFold2 (relatedly a description of the activation state of the AlphaFold2 structure may be useful). If the Y18:F309 interaction only happens when the activation loop is phosphorylated, is the autoinhibition by the PH domain only at the step of C-tail phosphorylation and does that interaction require an activation loop phosphorylation? Does the PH domain interaction happen equally as strongly with a variant in which the AL is unphosphorylated? In cells Y18A AKT lacks C-tail phosphorylation. As upstream kinases are responsible for this C-tail phosphorylation, how does the PH domain:Kinase domain interaction block C-tail phosphorylation?Reviewer #3 (Recommendations for the authors):1. Validation data that should be included in the intact MS data for all Akt constructs characterised in this manuscript, as well as the gel filtration traces, showing monodispersity for all constructs. The authors mention in the conclusion that they see phosphorylation of the turn motif, but no data is included here. Previous work has shown that the ligation strategy employed here leads to protein lacking turn motif phosphorylation , however, if this is the case here this may cause the protein to be less stable, however intact MS traces will also validate that there are no other differences between the generated proteins.2. The authors should alter their results and discussion to more clearly describe some of the uncertainty of their analysis of the alphafold model. At a minimum, the inclusion of the predicted aligned error, highlighting some of the uncertainty of the PH-kinase interface needs to be included. In addition, the results and Discussion section need to be rewritten to describe this point. The 309L mutant does not seem definitive, and molecular conclusions should be subdued. Essential revisions:There are a number of reviewer suggestions to further strengthen this manuscript (see specific reviewer comments below). In particular, two important issues are identified that the reviewers request be addressed in a revised manuscript:1) Questions are raised about the validity and predictive value of the AlphaFold model. Please see specific reviewer comments below and address them accordingly.We share the general concern that the confidence level in the AlphaFold predicted model of the autoinhibited Akt state is modest. However, as we mention, the experimentally determined crystal structures also have their limitations. We by no means believe that the AlphaFold model should be accepted uncritically but we are of the opinion that it is a publicly available resource for the community to inspect and analyze. As far as what the key templates were that drove the AlphaFold model, we are of course unsure, but there appear to be elements of the active kinase domain structure in the absence of the PH domain which date back to the early 2000s. However, for the phospho-Thr308 state, the form used in our studies, we think that an active kinase domain conformation is reasonable. In any event, we used the AlphaFold structure for hypothesis generation, namely to examine a possible unexpected interaction between Tyr18 and Phe309. The experimental data suggest that this interaction is plausible, although by no means definitively proven. We now summarize these points in the revised manuscript.2) Further validation of protein samples is requested. Specifically, MS quantification and gel filtration profiles are requested.In addition, the framing of this work creates some confusion. Given that this is submitted as a Research Advance, it is framed in the context of different phosphorylations of the C-tail on AKT. Since the results then focus on side chains in the PH domain the framing, as presented, creates some confusion about the goals at the outset and the eventual findings. The reviewers are confident that the authors can reframe the work in a manner that both explains the connection to earlier findings and clearly steers the reader through the present work.We now incorporate the requested mass spectrometry and size exclusion chromatography data in the revised manuscript. The mass spec data support that the composition of the semisynthetic Akt proteins prepared by expressed protein ligation are consistent with their proposed structures, taking into account some heterogeneity due to linker phosphorylations as reported previously . The SEC profiles show that these Akt proteins appear monomeric and monodisperse.Reviewer #1 (Recommendations for the authors):The following are provided to improve the quality of this manuscript:1) Figure 3A seems to show multiple peaks for the wt protein possibly indicative of slow exchange (the mutant spectrum appears greatly simplified). The authors do not adequately comment on this or attempt to establish whether the resonances that appear to give rise to multiple peaks are structurally related. Moreover, it is unclear whether the wt sample could be subject to changes in temperature or ZZ-exchange experiments to more carefully analyze the possibility of multiple species in slow exchange. This seems relevant to the idea that the R86A mutation 'stabilizes' the PH domain and in so doing pre-organizes the PH domain for binding to the kinase domain.1H-15N HSQC led us to the conclusion that there was a single conformation. To illustrate our thought process here, we present We thank the Reviewer for carefully looking at our spectra and proposing this hypothesis. The WT isolated PH domain is not stable and crashes out of solution in a few hours despite of our attempts to find conditions to stabilize the protein. This impedes the possibility of performing long NMR experiments, including relaxation dispersion, CEST and ZZ exchange experiments that report on multiple conformations. Our initial analysis based on the number of signals we observed in the 1H-15N HSQC spectra recorded at different temperatures. As the reviewer suggested we observed multiple resonances for certain signals at 20 °C, which collapses to a single signal at lower temperatures. When residues corresponding to these signals are plotted on the structure, they cluster in the general vicinity of the R86A site. We further investigated the potential existence of multiple conformations in the R86A mutant and saw no evidence for it from the 1H-15N HSQC. We have recorded relaxation dispersion experiments on the stable R86A sample and do not observe evidence for exchange on the µs-ms time scale.However, upon the insightful comment of the reviewer we went back to the data and analyzed the Thanks to the critical observation of the Reviewer, we now added the following section of text to the manuscript and a new extended figure in support of the statement.1H-15N HSQC spectrum  supports this hypothesis. A few resonances present a secondary, weaker, signal . These weak signals could be due to a minor conformation of the same residue or could correspond to an unassigned residue. We investigated these weak resonances as function of temperature and observed that they collapse into a single signal at lower temperatures, supporting the case of multiple exchanging conformations. Furthermore, the residues that display multiple conformations cluster around R86 in a disk-like arrangement reminiscent of the CSPs between WT and R86A . Taken together with its lower stability and melting temperature, this collectively suggests the possibility of multiple exchanging conformations in WT PH domain. However, the limited stability of the sample impedes further investigation by NMR thus we cannot fully confirm the presense of multiple conformations.“Though the X-Ray structures of the WT and R86A PH domains are very similar, the NMR chemical shift perturbations at locations distal to the mutation site could be explained by changes in intrinsic dynamics. One observation from the WT PH domain 1H-15N HSQC spectrum of the R86A PH domain does not contain these weak, secondary, signals  which suggest that R86A mutation removes some of the WT dynamics. The improved stability of R86A PH domain allowed us to conduct relaxation-dispersion experiments that further confirmed the absence of dynamics on the µs-ms timescale . We went on and measured the R1, R2, Etaxy relaxation rates and heteronuclear Overheauser effect (hetNOE) for the R86A mutant . These relaxation rates show high rigidity . Together with the observed high stability and melting temperature of R86A, this proves the absence of multiple exchanging conformations in the R86A PH domain.It should be noted that the Thus there is a strong possibility that the WT PH domain, in contrast to the R86A mutant, is significantly more dynamic and that this could be the reason for the significant chemical shift perturbations. These results may suggest that the PH domain conformation of the isolated R86A PH domain is preorganized before binding to the kinase domain to effectively autoinhibit Akt.”2) The notion that the R86A mutation stabilizes the PH domain seems to be based on the longevity of the protein sample. Could a more direct measure of protein stability that does not require the long acquisition times of the NMR relaxation experiments  be used to support this idea?We are grateful to the Reviewer for this suggestion. We have performed thermal shift differential scanning fluorimetry on WT and R86A PH domains. New Figure 3 —figure supplement 1 shows that the melting temperature of R86A is significantly higher (51°C) than that of WT (42°C).We added the following sentence to the manuscript:“Differential scanning fluorimetry revealed that R86A has increased melting temperature (51+/-1 °C) compared to WT PH domain .”3) In describing the data for Figure 3—figure supplement 3, the authors state that "we observe similar patterns of CSPs and peak broadening indicative of the interaction of the PH domain with the rest of the protein. Though the comparative CSP patterns of the R86A and WT PH domains when anchored to the rest of Akt look similar, there are subtle differences…". I agree with this conclusion but due to the absence of many peaks in the wt spectrum it is hard to pick out the similarities between the WT and R86A mutant as presented – in fact the histograms in Figure 3—figure supplement 3 look quite different. Perhaps the language used to compare the two datasets can be modified to make the point clearly without creating confusion. Importantly, it seems the authors need to clearly show the differences around residues 67-73.We agree that the phrasing of the CSPs interpretation could create confusion. We sought to clarify this and draw three main conclusions from our data: (i) the affinity of R86A for the kinase domain is higher than WT, resulting in CSPs of higher magnitude, (ii) the R86A CSP pattern is still consistent with our interaction model between PH and kinase domains and (iii) there are however small differences in areas where we did not expect interaction.The manuscript now reads:“The average CSPs on the PH domain in the context of FL Akt are higher in the case of the R86A mutant in line with the higher binding affinity. In both cases, we observe similar patterns of CSPs and peak broadening indicative of a similar interaction of the PH domain with the rest of the protein. Specifically, aa 14-22 show high CSPs or peak broadening in both cases and this region is expected to be in the core of the binding interface to the kinase domain and aa81-90 similarly show indications of binding, as expected from our previous model . Though the comparative CSP patterns of the R86A and WT PH domains when anchored to the rest of Akt look similar, there are subtle differences around residues 67-73  which indicates that the R86A mutation might reorganize the interaction surface and/or affinity.”We made a new Figure 3 —figure supplement 5C that specifically shows the CSPs of Arg67, Arg69 and Asn71.4) With regard to residues 67-73 it is intriguing that this is where the CSPs seem to differ between wt and R86A. Inspection of the structure shows that R69 in this region is involved in very close interactions with W22 (cation π interaction?). As well, R15 sits on the opposite face of W22 and both R15 and W22 surround the important E17-Y18 segment. Moreover, R69 is surrounded by prolines – could proline isomerization be at play here? Certainly, this is an event that could induce the slow exchange that is suggested in the 1H-15N HSQC spectrum of wt .We thank the Reviewer for suggesting this model. It is interesting that these differences in the 67-73 segment arise only in the context of full-length Akt, neither CSPs nor crystal structure show significant differences in this region in isolated PH domains. Unfortunately, the resonances corresponding to Arg15 and Trp22 are broadened beyond detection in full-length Akt spectra, which makes it difficult to assess the model further.The Pro isomerization inducing multiple conformations in an interesting idea. In response to this question, we looked at the CB shifts of Pro68 and Pro70 in R86A which are 31.9 ppm and 32.8 ppm, respectively, suggesting trans conformations. The poor quality of HNCACB for WT did not allow CB assignments for these Prolines. We do not see clear indications of multiple conformations for the neighboring Arg67, Arg69 or Asn71 in the spectra of isolated WT or R86A PH domains. The residues in the vicinity of the two prolines are broadened beyond detection in the R86A fulllength Akt spectrum preventing further analysis. Hence, our data can neither strongly support nor negate this hypothesis. It should be noted that both these Pro residues are in trans conformations in the crystal structures of the PH domain, both for WT and R86A mutant.5) In Figure 7A-D.  "PH domain" in blue is the R86A mutant? If so please clarify in the figure and caption – the text indicates the R86A PH domain while the figure is unclear. Assuming Figure 7 is the R86A mutant, was the corresponding data for the wt PH domain also acquired to assess conformational heterogeneity at the Y18 position for wt versus R86A mutant? Such an experiment could help better understand the effects of the R86A mutation on the Y18 sidechain.We apologize for the lack of clarity in this section and thank the reviewer for pointing it out. Yes, we used the R86A mutant for this experiment to enhance the affinity of the interaction between the PH and kinase domains of Akt. Moreover, the inclusion of R86A mutation in these constructs was helpful for their stability while performing NMR studies. We have now clarified this in the caption for Figure 7A-D. The corresponding data for the WT PH domain would certainly be nice to have. However, the stability and expression level of the WT PH containing 19F-Tyr18 was poor rendering the semisynthesis of full-length 19F-Tyr Akt impractical. Thus, for assessing the effects of the F309L mutation and phosphorylation of T308 on the Y18 sidechain, R86A mutation was included in each of the constructs used in this set of experiments.Reviewer #2 (Recommendations for the authors):The multi-pronged approach systematically investigates the interaction between PH:Kinase, stability/structure of PH domain, activity of AKT and half-life of activation loop phosphorylation. Methodologically this work is a tour de force with the use of 3-way ligation to generate specific, stoichiometric post-translationally modified variants to study and highlights the limitations of studies using non-physiological variants.This paper is written for an AKT expert and may not successfully connect with the broader eLife audience. The main conclusion of the paper comes quite late and was unanticipated. While such findings are exciting, I found it hard to connect the earlier and later results into a coherent finding.– This paper is written for an expert and as such may not successfully connect with the broader eLife audience. For example, an introduction to the regions of the PH domain responsible for lipid binding and those proposed to bind AKT would help provide context for understanding the early mutagenesis. Perhaps figure 1B could be relabeled to identify these surfaces of the PH domain? Similarly, introducing the models of AKT autoinhibition earlier would help provide a framework for the subsequent experiments.We thank the Reviewer for the recommendation to highlight regions of the PH domain involved in PIP3 binding and surfaces proposed to interact with the kinase domain. We also agree that introducing some of the details about the prior models for autoinhibition earlier makes sense. We have made these edits in the revised manuscript.– The paper is framed as a study in the role of different phosphorylations of the C-tail on AKT but ends with a result that is separate from the tail. At the end of the paper, I'm still pondering how the tail could/would influence this new autoinhibitory interaction. Can the authors clarify how the phosphorylation of the AKT tail influences interactions/activities of variants tested and how those states would influence their model? Relatedly, several times throughout the paper I struggled to determine whether the variant used had the tail present and, if so, its phospho-state. I'm still not sure if the experiments in Figure 2C/Figure 4A were performed with or without the tail as there is a disconnect between the carton and description. Also, the description of the variants I think varies-- is "non-C-tail phosphorylated Akt" the same as" full-length AKT mutations in the absence of C-tail modification"?The Reviewer is correct that we have not resolved the details of how phosphorylation of Ser477/Thr479 can activate. We think that it is possible that these phosphoSer/Thr directly bind Arg86 but have no direct evidence of this. We have added a bit more of this to the Discussion. Regarding which Akt forms were used in a particular experiment, we apologize for the confusion. We have added cartoons like in Chu et al. 2020 to clarify in the revised manuscript . Indeed "non-C-tail phosphorylated Akt" is the same as " full-length AKT (mutations) in the absence of C-tail modification" in this manuscript.– Earlier work from this lab suggests that the linker between the PH and kinase domain influences inhibition. As this region was missing in experiments using the PH domain in trans, could the authors speculate on how the lack of the linker would influence those results?The Reviewer astutely points out the potential that “in trans” interaction may not recapitulate precisely the intramolecular interaction between kinase and PH domains. As they note, our prior enzymatic studies suggest that linker length and composition influence the PH-kinase intramolecular interaction. Therefore, the intermolecular (in trans) interaction experiments should be interpreted cautiously. Having said that, the intermolecular affinity interactions associated with R86A, Y18A, and E17K correlated fairly well with the full-length semisynthetic enzymatic activities of the Akt forms, suggesting that the “in trans” experiments can capture at least some aspects of the intramolecular recognition.– Arguments that the PH domain of R86A retains the same fold as WT but is more stable/less dynamic are indirectly and weakly supported. While this claim is not critical to the later arguments, the data presented are not compelling. The authors claim that the WT PH domain was less stable b/c it degraded during the NMR experiments but do not show data supporting that claim. They then conclude the structures are only slightly changed between WT and R86A based on the ~60% of residues they can assign. If the WT PH domain is degrading during the course of the experiment, how legitimate are the assignments from this experiment and how do they know they are looking at a degraded protein? The analysis of the B-factor of the crystal structure also does not provide much insight into dynamics, especially without any comparison to the b-factor distribution of the WT PH domain – A quick look in pymol reveals a similar pattern of B-facto distributions for the WT PH Domain. Further in 7MYX the loop 42-49 is disordered but is well resolved in the WT structure (1UNP). Since Chu 2020 eLife says this loop is important for binding C-tail and PIP3 – what significance does the additional disorder mean? This disordered loop also implies the statement that the two structures of PH domains are in "high concordance" and "identical" is an overstatement (pg7).1H-15N HSQC, which we did not observe. In addition, the resonances will shift as a result of the removal of residues, this was also not observed. Another possibility is that the protein aggregates over time, which we physically observe as precipitation. The aggregated and precipitated protein do not contribute signals in solution NMR. Our time course measurements show no change in peak position rather reduction in intensity, in line with protein aggregation. Therefore, we are confident of the NMR backbone assignments made for the WT and R86A PH domains although concede that the instability of the WT PH domain complicates structural comparisons by NMR. As recommended, we have softened the language in the revised manuscript on the concordance of the two structures given the limitations alluded to.Thanks for these comments. Regarding the stability of the R86A PH domain vs the WT PH domain, we have now augmented our prior NMR evidence with differential scanning fluorimetry as discussed above. The R86A has 9 °C increased thermal stability compared to WT . We did not observe changes in chemical shifts over the course of our NMR experiments, only reduction of the intensities of the peaks corresponding to the starting conformation. If the reviewer suggests the possibility that the WT PH domain is proteolytically degraded during the course of the experiment, we can conclusively state that this is not the case. In case of proteolytic degradation, we would see additional resonances in a specific part of the – Can the authors speculate at the precise step that the PH domain is inhibiting AKT? The Α Fold 2 structure looks to have the activation loop in the out (ie activated state) even though there is no phosphorylation on the activation loop for AlphaFold2 (relatedly a description of the activation state of the AlphaFold2 structure may be useful). If the Y18:F309 interaction only happens when the activation loop is phosphorylated, is the autoinhibition by the PH domain only at the step of C-tail phosphorylation and does that interaction require an activation loop phosphorylation? Does the PH domain interaction happen equally as strongly with a variant in which the AL is unphosphorylated? In cells Y18A AKT lacks C-tail phosphorylation. As upstream kinases are responsible for this C-tail phosphorylation, how does the PH domain:Kinase domain interaction block C-tail phosphorylation?It would be interesting to know what the affinity of the PH domain is for the nonThr308 phosphorylated kinase domain is, although this has not been measured. We will plan to do this in a future study. We believe that the importance of the PH domain’s inhibition of Akt is most clear when Thr308 is phosphorylated since the non-Thr308 phosphorylated form is intrinsically autoinhibited without the PH domain. We are uncertain about the timing of the Phe309 and Tyr18 interaction and there is insufficient data to make a confident statement about this, although our model is that this Phe309/Tyr18 interaction could occur in the presence of Thr308 phosphorylation. Future experiments can hopefully help address this.Reviewer #3 (Recommendations for the authors):1. Validation data that should be included in the intact MS data for all Akt constructs characterised in this manuscript, as well as the gel filtration traces, showing monodispersity for all constructs. The authors mention in the conclusion that they see phosphorylation of the turn motif, but no data is included here. Previous work has shown that the ligation strategy employed here leads to protein lacking turn motif phosphorylation , however, if this is the case here this may cause the protein to be less stable, however intact MS traces will also validate that there are no other differences between the generated proteins.These mass spectra and gel filtration profiles are now included in the revised manuscript for the key and representative semisynthetic proteins prepared in this study (the majority of those prepared). As reported recently, the mass spectra show that the correct masses are observed for the major expected species  with some heterogeneity indicating additional phosphorylations localized to the linker region. The SEC chromatograms show the position of the monomeric and monodisperse forms of the Akts isolated here.2. The authors should alter their results and discussion to more clearly describe some of the uncertainty of their analysis of the alphafold model. At a minimum, the inclusion of the predicted aligned error, highlighting some of the uncertainty of the PH-kinase interface needs to be included. In addition, the results and Discussion section need to be rewritten to describe this point. The 309L mutant does not seem definitive, and molecular conclusions should be subdued.We thank the Reviewer for this suggestion and have attempted to be clearer about the uncertainties around the AlphaFold model and the interpretation of the F309L data. Also we made a new Figure 6 – supplement figure 1 showing the AlphaFold-predicted full-length Akt structure with its confidence score (pLDDT).


The functional properties (water absorption capacity and water solubility index) of the flours were determined. Sensory evaluation of biscuits was determined. The proximate and mineral contents of the most preferred biscuits were determined. Results showed that the substitution significantly increases the functional properties of the flours (P < 0.05). Sample 75% (T3) is the most preferred biscuit. The incorporation of sweet potato and soy flour in the formulation significantly increases the moisture, fat, protein, fiber, copper, manganese, calcium, magnesium, and potassium contents of the biscuits (P < 0.05). Biscuit 75% (T3) records the highest levels in protein and fiber while sample T0 (control) records the highest levels of iron, zinc, and sodium. In terms of overall acceptability and nutrient contents, sample 75% (T3) is the best biscuits.The aim of this study was to evaluate the nutritional composition of biscuits from wheat-sweet potato-soybean composite flours. Substitutions of wheat flour with sweet potato and soybean flours at the rate of 25%  (T1), 50%  (T2), 75%  (T3), and 100%  (T4) were made to obtain the wheat-sweet potato-soybean composite flours. Wheat flour without sweet potato and soybean flour was used as control (T The consumption of wheat products especially bread and biscuits occupies a prominent place in the diet of populations, even in non-wheat-producing countries. The latter are becoming more and more dependent on nations of wheat producers, particularly during economic crises where wheat is very expensive to import. This cereal is imported while our local products remain unexploited and are decaying .World wheat production is around 580 million tons per year, of which 350 million tons are processed into flour and meal. In Cameroon, since the year 2000, the quantities of wheat imported have increased sharply. Until 2007, they were around 280,000 tons. From 2008, there was a significant increase in these quantities of which 681,778 tons were imported in 2017 at a cost of around 103.7 billion francs CFA with an average price of around US $300 per ton. The unit import price increased to 3.3%.In Cameroon, the attempt to introduce wheat cultivation has not produced the expected results. However, to reduce the use of imports of wheat flour and to improve the value of local products, the solution is to integrate the tubers (sweet potatoes and cassava) in manufacturing of bread wheat flour when producing biscuit. The tubers have not yet undergone a significant modern transformation, due to their low value. To remedy this situation, technologies are increasingly being developed to replace wheat flour with flour obtained from local food resources . Sweet pAlso, sweet potato is an important source of carbohydrates 96%), in the form of simple carbohydrates and dietary fibers, which plays an important role in energy deficiencies . Apart f%, in theIn Cameroon, the annual production of sweet potato is 288,970 tons and that of soya is 12,544 tons [Ipomoea batatas), soybean (Glycine max), and other ingredients were collected from the local market, Maroua, a town in the Far North region of Cameroon.Sweet potato , and dried in an oven at 105°C for 24 h. The dried pellet weight (M2) was determined. The water absorption capacity (WAC) was calculated as follows: WAC = (M1 − M2) × 100/M2. All samples were analyzed in triplicate.The water absorption capacity of the studied flours was determined according to the method of Beuchat . A samplM0 − M1)/M0 × 100, where M0 is the mass of sample and M1 is the final mass after dissolution in water. All samples were analyzed in triplicate.The water solubility index (WSI) was determined according to Anderson et al. . WSI refThe quantities of each ingredient used in the preparation are presented in After weighing the ingredients, the margarine and sugar were mixed using a manual thresher to give a cream. The beaten egg was incorporated into the cream, and then, the mixing continues until a snow-white paste was obtained.The ingredients and raw materials in powder form previously prepared  were added to the cream and the whole were manually mixed. Water was added until the dough was smooth. After kneading, the dough was spread on the pastry board using the rolling pin. The display thickness was fixed at 4 mm for better cooking. This thickness was measured using a fabricated frame.The oven was preheated to 180°C. The biscuit dough pieces were placed on the baking trays and placed in the oven at a temperature between 140 and 150°C. The temperature was set manually. This operation allowed us to obtain good quality biscuits by adjusting the baking temperature. When the biscuits are golden brown after 20 min, the trays are removed from the oven and cooled. After cooling, the biscuits were packaged in sachets and then closed with a heat sealer.The sensory evaluation was carried out in the University of Maroua. Thirty panelists aged between 18 and 35 years, all of them students of the University of Maroua, participated. They were informed about the objectives and asked not to eat 2 h before. The samples were compared on the basis of the following organoleptic characteristics: taste, color, texture, flavor, and overall acceptability. About 3 g to 5 g of each sample was served to each of the panelists under a white light in a random order. An information sheet was given to them. Between each tasting, they had to rinse their mouths with mineral water and pause for about 2 min in order to avoid appreciation errors. Each attribute was scored based on its intensity scaled on a 9-point hedonic scale .The proximate composition of the flours and biscuits was determined according the standard analytical methods (AOAC) . All samThe method described by AOAC  was usedP < 0.05 using Duncan multiple range test.Data were presented as means ± standard deviation (SD). Values were statistically analyzed by one-way analysis of variance (ANOVA test) using IBM SPSS Statistics version 20.0.1 software package, Armonk, New York, USA. Differences were considered significant at P < 0.05). The moisture content is a crucial parameter in the storage of flour. Indeed, a moisture content of flour greater than 12% promotes the development of microorganisms. The lower the moisture content of the flour, the more it is possible to hydrate it during kneading to reach an optimal consistency of the dough [The moisture content in the flours ranged from 7.49 (soy bean flour) to 10.58 g/100 g (wheat flour) and was significantly different (he dough .P < 0.05). The ash content is an indicator of the purity of the flour. It is related to its extraction rate and the mineralization of the grains milled. It also defines the commercial types of flour, particularly with regard to wheat flour [The ash content in the flours was in the range of 0.99 (wheat flour) to 2.01/100 g (soy bean flour) and was significantly different (at flour .P < 0.05). The fat content obtained for sweet potato flour is lower than (1.54%) obtained by Siédogo [The fat content in the biscuits ranged from 0.55 for sweet potato flour to 27.3/100 g for soy bean flour and was significantly different ( Siédogo . This di Siédogo .P < 0.05). The protein content of wheat flour (13.48%) is greater than 7%, corresponding to the minimum set by the Codex Alimentarius [Protein content in the biscuits varied from 2.57 for sweet potato flour to 36.44 g/100 g for soy bean flour and was significantly different (entarius . The proP < 0.05). The high fiber content in sweet potato and soy flour may increase the water absorption capacity of the flour.The fiber content in the biscuits was in the range of 0.56 to 5.01 g/100 g. The lowest fiber content was obtained from wheat flour while the highest was obtained from soy bean flour. The values of fiber content were significantly different (P < 0.05). Energy content in the biscuits varied from 348.31 for sweet potato to 478.43/100 g for soy bean flour and was significantly different (P < 0.05).Carbohydrate content in the biscuits varied from 21.74 for soy bean flour to 83.27 g/100 g for sweet potato flour and was significantly different (0) to a high value 84.33% in T4 (100%). Substitution significantly increases the water absorption capacity of the flours (P < 0.05). The increase in water absorption capacity would therefore be due to the availability of hydrophilic carbohydrates and functional groups of proteins in flour which are capable of forming hydrogen bonds with water. Indeed, the water absorption capacity depends on the affinity of flour with water. The higher the affinity, the higher the water absorption capacity.The water absorption capacity of the flours ranged from a low value 73% in control (T0) and 42.76% of T4 (100%). Substitution also significantly increases the water solubility index of the flours (P < 0.05). These results are in agreement with those of Legesse and Admassu [The water solubility index of the flours varied between 17.31 of control  were found to be the best and biscuit T2 (50%) was closer to biscuit T3 (75%). The taste is the primary factor which determines the acceptability of any product, which has the highest impact as far as market success of product is concerned. Biscuit containing 100% soy and sweet potato flours (T4) was rated the poorest in taste (6.26). Biscuit containing 75% soy and sweet potato flours (T3) has the highest mean score (7.61). The mean scores for color of the biscuits changed from 5.4 to 7.19. The highest score (7.19) was also obtained in biscuit T3 (75%). As increase in soy flour, mean score for color was also increasing. This is in contradiction with the result of Banureka and Mahendran .The texture of the crust was related to the external appearance of the biscuit top which implies smoothness or roughness of the crust. The highest score (7.41) was also obtained in biscuit T3 (75%). Except in biscuit T1 (25%) and biscuit T4 (100%), substitution increases the texture of biscuits. In the case of flavor of the biscuits, it was decreased with an increase in the substitution except in biscuit T2 (50%) and T3 (75%). Overall acceptability includes many implications, which is an important parameter in organoleptic estimation. Biscuit T3 (75%) had the highest mean value (7.61), and T1 (25%) had the least mean value (6.23) for the overall acceptability.P < 0.05) and varied from 8.62 for T0 (control) to 9.59/100 g for T3 (75%). We then note that the incorporation of sweet potato and soy flour in the formulation significantly increases the moisture content. This increase would be the direct consequence of the water absorption capacity of the flours which we have used. Indeed, the functional properties of the compound flours presented above in the characterization of the flours show that the flours composed of wheat-sweet potato-soybeans have a high capacity for absorbing water compared to control and this capacity increases with the substitution.The moisture content in the biscuits significantly increased with substitution (P < 0.05) and varied from 1.36 of biscuit T2 (50%) to 2.02 g/100 g of biscuit T3 (75%). The values obtained are lower compared to the value reported by Eyenga et al. [P < 0.05) and varied from 2.02 for biscuit T0 (control) to 2.55 g/100 g for biscuit T3 (75%). The values obtained were compared to those found by Siedogo [The ash content in the biscuits also significantly increased with substitution  and varied from 11.41 for biscuit T0 (control) to 13.08 g/100 g for biscuit T3 (75%). This increase could be due to the increase in the proportion of soy flour. The values obtained are higher compared to the value reported by Eyenga et al. [P < 0.05) and varied significantly from 1.4 to 4.2 g/100 g. The lowest fiber content was obtained from biscuit T0 (control) while the highest was obtained from biscuit T3 (75%). Similar trends in fiber increase in fiber content were also reported by Ayo et al. [Protein content in the biscuits significantly increased with substitution . The highest carbohydrate content was observed in biscuit T0 (control) (75.02 g/100 g), and the lowest was reported in biscuit T3 (75%) (68.90 g/100 g). A similar trend in decrease in carbohydrate content was also reported by Ayo et al. [P < 0.05) and varied from 349.4 for biscuit T3 (75%) to 363.9 kcal/100 g for biscuit T0 (control). Those values are lower compared to 506 kcal/100 g found by Eyenga et al. [Carbohydrate content in biscuit significantly decreased with substitution  and ranged from 0.302 for biscuit T0 (control) to 0.374 mg/100 g for biscuit T3 (75%). The copper content increases with substitution. Copper participates in the activity of many enzymes and chemical reactions. It is involved in the oxidation of glucose and is essential for the proper functioning of the myocardium (muscle of the heart). Copper controls bone mineralization and the quality of cartilage and plays a role in the regulation of neurotransmitters, by regulating mood, sleep, memory, and attention. Copper is also involved in the immune process and iron metabolism [The copper content in the biscuits significantly increased with substitution (tabolism . The conP < 0.05) and ranged from 0.908 for biscuit T0 (control) to 0.9824 mg/100 g for biscuit T3 (75%). The results obtained are higher compared to those reported by Bello et al. [The manganese content in the biscuits also significantly increased with substitution . Biscuit T0 (control) records the highest content (5.89 mg/100 g), and biscuit T3 (75%) records the lowest score (3.456 mg/100 g). The results of this study are comparable to those found by Maria et al. [The iron content in biscuits significantly decreases with substitution . Biscuit T0 (control) records the highest content (2.127 mg/100 g), and biscuit T3 (75%) records the lowest score (1.509 mg/100 g). The zinc contents are close to those found by Campos-vega [0 (control) is the best source of zinc.The zinc content also decreases significantly with substitution (pos-vega  (1.4-1.9pos-vega . Among tP < 0.05) and varied from 30 for biscuit T0 (control) from 77 mg/100 g for biscuit T3 (75%). The increase in calcium content with substitution can be explained by the presence of soybeans which 277 mg calcium/100 g [The calcium content in the biscuits also significantly increased with substitution  and ranged from 32 for biscuit T0 (control) to 49 mg/100 g for biscuit T3 (75%). The results obtained are low compared to 62 mg/100 g obtained by Maria et al. [The magnesium content in the biscuits also significantly increased with substitution  and ranged from 184 for biscuit T0 (control) to 541 mg/100 g for biscuit T3 (75%). The magnesium content also increases with substitution. The presence of soy and sweet potato in the formulation is responsible for this increase in fact; Badila et al. [The potassium content in the biscuits also significantly increased with substitution  and ranged from 94 mg for biscuit T3 (75%) to 254 mg/100 g for biscuit T0 (control). A low intake of sodium and sufficient potassium constitutes the dietary challenge that humans have to face daily. High consumption of dietary sodium plays an important role in raising blood pressure [The sodium content in the biscuits significantly decreased with substitution (pressure . The respressure . Therefo0 (control) would meet 88.83%, 75.67%, 84.15%, 70.9%, 6%, 40%, 6.13%, and 25.4% of the dietary reference intakes, respectively, for copper, manganese, iron, zinc, calcium, magnesium, potassium, and sodium. Consumption of 100 g of biscuit T2 (50%) would meet 96.17%, 79.58%, 51.4%, 56.83%, 7.4%, 50%, 14.46%, and 24.1% of the dietary reference intakes, respectively, for copper, manganese, iron, zinc, calcium, magnesium, potassium, and sodium. Consumption of 100 g of biscuit T3 (75%) would meet 110%, 81.83%, 49.37%, 50.3%, 15.4%, 61.25%, 18.03%, and 9.4% of the dietary reference intakes, respectively, for copper, manganese, iron, zinc, calcium, magnesium, potassium, and sodium. The consumption these biscuits will significantly contribute for improving the RDI of the children.The required daily intake (RDI) values for minerals in foods intended to human consumption were established by the United States Department of Agriculture . The RDI0 (control) records the highest levels of iron, zinc, and sodium. In terms of overall acceptability and nutrient content, biscuit T3 (75%) is the best biscuit. The biscuits produced from the mixture wheat flour-sweet potato-soy would be beneficial for reducing protein and mineral deficiencies.Substitution significantly increases the water absorption capacity and the water solubility index of the flours. Biscuit T3 (75%) is the most preferred biscuits. The incorporation of sweet potato and soy flour in the formulation significantly increases the moisture, fat, protein, fiber, copper, manganese, calcium, magnesium, and potassium content in the biscuits. Biscuit T3% (75%) records the highest levels in protein and fiber while biscuit T