Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity.The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a  Plasmodium falciparum malaria is estimated at 216 million cases annually, with approximately 500,000 deaths occurring worldwide [P. falciparum vaccine RTS,S (MosquirixTM) is based on the pre-erythrocytic stage of the parasite life cycle and is the only malaria vaccine to have completed phase III clinical trials [Mortality caused by orldwide . The mosl trials  and is cl trials . To achiP. falciparum. The progress of sexual-stage antigens as vaccine candidates is further limited, in part, by the difficulty to express high yields of properly folded recombinant antigens, and the requirement for vaccine approaches that generate high and sustained levels of antibodies for effective transmission-blocking activity. Leading vaccine candidates that are expressed during the P. falciparum sexual-stage include Pfs230 and Pfs25. Pfs230 is expressed on the surface of gametocytes that reside within the human host, while Pfs25 is expressed on the surface of female gametes, zygotes and ookinetes in the mosquito vector [Transmission-blocking vaccines will need to induce a potent antibody response within the host to inhibit the downstream development of parasites in the mosquito vector after a blood meal . This wio vector . Numerouo vector ). Therefo vector –12, and o vector –13, demoThe development of potential vaccines requires the induction of strong and sustained immune responses in humans, which has been challenging to achieve. The classic approach of a subunit vaccine includes the delivery of a recombinant antigen formulated with the appropriate adjuvant (reviewed in ). ExpresP. falciparum sexual-stage antigens, Pfs25 and the domains Pfs230c [Hansenula polymorpha [Here, we describe a novel platform that incorporates sexual-stage malaria vaccine candidates, Pfs230 and Pfs25, into a VLP scaffold that is based on the duck hepatitis B virus (DHBV) small surface protein (dS). In contrast to the human HBV platform, the DHBV delivery platform allows the expression of large and complex proteins , which a Pfs230c  derived lymorpha . The sucHansenula polymorpha . Pfs230c-dS consists of amino acids (aa) 443–1132 (630 aa fragment) from full-length Pfs230 [Hansenula-derived yeast cell line. The purification of the chimeric VLPs was based on a downstream process approved for human VLP vaccine production from yeast [P. falciparum antigens [The production and purification of the three different kinds of chimeric VLPs  was conducted as previously described ,25. Geneh Pfs230 . The fusP. pastoris termed Pfs230D1H [The expression of monomeric recombinant Pfs230D1M was performed in HEK293F cells as previously described . Brieflyfs230D1H . To assefs230D1H .Chimeric VLPs or monomeric recombinant proteins were coated onto Maxisorp microtiter plates (Nunc) at 1μg/ml in PBS and incubated overnight at 4°C. Plates were blocked with 1% casein in PBS (Sigma-Aldrich) for 2h at 37°C before primary antibodies were added . Secondary HRP-conjugated antibodies  were used to detect antibody binding. Colour detection was developed using ABTS or TMB liquid substrate (Sigma-Aldrich), which was subsequently stopped using 1% SDS (for ABTS) or 1M sulphuric acid (for TMB). PBS was used as a negative control and plates were washed thrice using PBS with 0.05% Tween in between antibody incubation steps. The level of antibody binding was measured as optical density at 405nm (for ABTS) or 450nm (for TMB).Antibody affinity was assessed using standard ELISA, with the addition of an antibody dissociation step using increasing concentrations of ammonium thiocynate (in PBS). This was incubated for 20 min at room temperature, following incubation with antibody samples . Colour detection was developed using ABTS .Mature stage V gametocyte-infected erythrocytes were generated as previously described ,29 and tThin blood smears of stage V 3D7 gametocyte-infected erythrocytes were fixed in 90% acetone and 10% methanol for 5 min at -20°C as previously described . BrieflyPfs230c-dS/dS and Pfs230D1M-dS/dS VLPs were formulated with equal volumes of sterile Alhydrogel aluminium hydroxide vaccine adjuvant  and incubated on the shaker for 5 min prior to immunisations. New Zealand White rabbits were immunised 2 weeks apart with 3 doses of chimeric VLPs. The total amount of protein and adjuvant used in each immunisation is presented in P. falciparum NF54 line were mixed with purified test IgG at 7.5 mg/ml, and the final mixture, which contained human complement, was immediately fed to ~50 female Anopheles stephensi mosquitoes through a membrane-feeding apparatus. Mosquitoes were kept for 8 days and dissected to enumerate the oocysts in the midgut . As the negative controls, total IgG purified from pre-immune rabbit sera were utilised. Only midguts from mosquitoes with any eggs in their ovaries at the time of dissection were analyzed. The human serum and red blood cells used for the SMFA were purchased from Interstate Blood Bank .IgG purification from individual rabbit serum samples was performed using Protein G columns  according to the manufacturer instructions and adjusted to a final concentration of 20 mg/ml in PBS. The standardized methodology for performing the standard membrane feeding assays (SMFA) was described previously . Brieflyp-values from single or multiple feeds were calculated using a zero-inflated negative binomial random effects model (ZINB model) described previously [p-values <0.05 were considered significant.The best estimate of % inhibition in oocyst density , the 95% confidence intervals (95%CI), and eviously . All staP. falciparum antigens, Pfs230 and Pfs25, were produced and purified as described [Chimeric VLPs expressing sexual-stage escribed ) which iTo understand whether these chimeric VLPs displaying sexual-stage antigens were capable of eliciting an immune response, the VLP constructs were used to immunise rabbits . Serum from immunised rabbits had significant antibody recognition of monomeric recombinant Pfs230D1M  or Pfs25We evaluated whether rabbit antibodies generated against Pfs230c-dS/dS VLPs were capable of recognizing native antigens expressed on the surface of mature stage V gametocytes by flow cytometry . The majTo address the functional significance of rabbit antibodies generated against sexual-stage chimeric VLPs, we examined the ability of these antibodies to inhibit mosquito infection through standard membrane feeding assays (SMFA) in the presence of human complement . FunctioOur work describes the immunogenicity of transmission-blocking malaria vaccine candidates presented on the surface of VLPs using a novel platform based on the duck hepatitis B virus. Leading sexual-stage antigens, Pfs230 and Pfs25 were engineered into chimeric VLPs and their surface expression characterised using specific antibodies. Data is presented in the accompanying manuscript [A conformational-dependent monoclonal antibody, 4B7, was used to characterise the display of antigen expressed by the Pfs25-dS/dS VLP. There were high levels of antibody recognition measured by ELISA to the Pfs25-dS/dS VLP, which correlated with the coating concentration of monomeric recombinant Pfs25. Similarly, using a Pfs230-specific polyclonal antibody, the display of antigen expressed by the Pfs230c-dS/dS and Pfs230D1M-dS/dS VLP was confirmed. This suggests that the Pfs25 and Pfs230 constructs had successfully been incorporated as fusion proteins into the chimeric VLPs. The incorporation rate of Pfs25-dS into VLPs was relatively low and optimization may be needed to increase incorporation rates prior to further evaluation of Pfs25 VLPs using this platform.Our immunogenicity studies showed that the chimeric VLPs could elicit an immune response in rabbits. For immunisations with Pfs230c-dS/dS VLP, two dosing regimens were used in the presence and absence of Alhydrogel. As the higher dose appeared to induce a better antibody response in rabbits, and formulation with Alhydrogel was needed for high antibody levels, immunisations with Pfs230D1M-dS/dS VLP were done at a single dose (using the higher dose) and formulated with Alhydrogel. Pfs230D1M-dS/dS VLPs were also formulated with Freund’s adjuvant for comparison. VLPs formulated with Alhydrogel induced substantial antibody responses, but responses were higher with Freund’s adjuvant. This was expected because Freund’s is a more potent adjuvant than Alhydrogel. Our results suggest that investigating other clinically-acceptable adjuvants that may induce higher antibody responses than Alhydrogel would be valuable. Further, rabbits were immunised with Pfs25-dS/dS VLP using only Freund’s adjuvant as a proof-of-principle to evaluate immunogenicity and transmission-blocking activity. Antibodies generated against the Pfs25-dS/dS VLP recognised monomeric recombinant Pfs25 by ELISA. Together, these findings suggest that immunisation with chimeric VLPs induced a good immune response in rabbits, further supporting the potential for the use of this VLP platform to present malaria antigens for vaccination. However, further detailed studies are needed to evaluate the immunogenicity and functional activity of antibodies generated by antigens presented as VLPs compared to monomeric recombinant proteins. In addition, we measured the ability of rabbit antibodies to recognise the surface of native gametocytes by flow cytometry. We found that rabbit antibodies generated against Pfs230c-dS/dS VLPs were capable of recognising the surface of mature, stage V gametocytes. This was also confirmed by immunofluorescence microscopy using fixed smears of mature gametocytes.Functional antibody responses were evaluated using standard membrane feeding assays, which are widely used to assess transmission-reducing activity . AntibodFurther, the Pfs230D1M construct is currently undergoing phase I clinical trials. Together, these findings support the importance of Pfs230D1M in the induction of functional transmission-reducing antibodies. Of note, transmission-reducing activity was not observed for antibodies generated against Pfs230c-dS/dS VLPs. This could have been due to the sub-optimal folding of Pfs230c, such that key functional epitopes were not displayed, or important antibody-binding epitopes were masked. These factors potentially resulted in low purity and reduced yield of chimeric VLP and thus led to the lack of functional antibodies generated. Interestingly, there was no clear relationship between IgG reactivity levels quantified by ELISA and transmission-blocking activity in SMFA. This was particularly evident for Pfs230D1M-dS/dS; immunization with VLPs formulated with Freund’s adjuvant generated higher IgG reactivity than using alum as the adjuvant, but IgG generated using alum tended to have greater activity in SMFA. There was no substantial difference in avidity between IgG induced using different adjuvants, which suggests the differences in transmission-blocking activity are most likely explained by differences in epitope-specificity of IgG. Future studies with a larger number of animals and immunization regimens may help further understand this.In conclusion, we have demonstrated the successful display of sexual-stage antigens Pfs230 and Pfs25 on the surface of chimeric VLPs. We have established a proof-of-concept which showed that these VLPs generated significant immune responses that recognised homologous recombinant protein and native sexual-stage antigens expressed on gametocytes. Further, these antibodies had the ability to block the transmission of parasites to mosquitoes through membrane feeding assays. Together, our results support the further evaluation of chimeric VLPs as a novel delivery platform for leading malaria vaccine candidates. Future studies to optimise antigen incorporation and presentation in VLPs and to evaluate different adjuvants and dosing regimens for immunization will further inform the potential utility of this strategy for malaria vaccine development.Conceptualization, JC, DW, LR, PG, JR, DA, MS, MP, and JB; Methodology, JC, DW, LR, DD, VJ, KM, TT and MP; Investigation, JC, DW, LR, DD and KM; Administration, VJ, JR, DA, MP and JB; Manuscript preparation, JC, DW and JB ; Funding Acquisition, JB and MP. Supervision, JB, MP, CL, MB. All authors approved the final manuscript.S1 Checklist(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 FigThe avidity of antibody binding to monomeric recombinant Pfs230D1M was assessed in serum from rabbits immunised with Pfs230D1M-dS/dS VLPs. The affinity of rabbit antibodies to bind monomeric recombinant Pfs230D1M was similar, irrespective of adjuvant formulation used for rabbit immunisations. Rabbits R1917 and R1918 received Pfs230D1M-dS/dS VLPs formulated with Freund’s adjuvant (Fre), while R1919 and R1920 received Pfs230D1M-dS/dS VLPs formulated with Alhydrogel (Alh). Antibody affinity was assessed by the dissociation of antibodies using increasing concentrations of ammonium thiocynate. Antibody binding is defined as the OD of ammonium thiocynate-treated samples/OD of untreated samples x100.(EPS)Click here for additional data file.


We examine the effect of different ligands on the human sGC HNOX domain. HNOX systems with gaseous ligands were generated and explored using Molecular Dynamics (MD). The distance between heme Fe2+ and histidine in the NO-ligated HNOX (NO-HNOX) system is larger compared to the O2, CO systems. NO-HNOX rapidly adopts the conformation of the five-group metal coordination system. Loops α, β, γ and helix-f exhibit increased mobility and different hydrogen bond networks in NO-HNOX compared to the other systems. The removal of His from the Fe coordination sphere in NO-HNOX is assisted by interaction of the imidazole ring with the surrounding residues which in turn leads to the release of signaling helix-f and activation of the sGC enzyme. Insights into the conformational dynamics of a human sGC HNOX domain, especially for regions which are functionally critical for signal transduction, are valuable in the understanding of cardiovascular diseases.Soluble guanylate cyclase (sGC) regulates numerous physiological processes. The β subunit Heme Nitric Oxide/Oxygen (HNOX) domain makes this protein sensitive to small gaseous ligands. The structural basis of the activation mechanism of sGC under the influence of ligands (NO, O These e, and NO . It is t, and NO ,6. The c2 bind with the heme group on the so-called distal site, while on the proximal side a coordinate bond forms between heme and histidine 105 (H105) (6c) [The crystal structure of the bacterial HNOX domain has been resolved, but essential heme binding pocket residues  are also conserved in the human HNOX [05) (6c) . The hem05) (6c) ,12,13.2+ coordination state of the HNOX heme favors the binding of small gaseous ligands, such as NO, O2, and CO [2. Discrimination between these diatomic molecules is controlled by heme distal site residues, whereas residues surrounding the proximal histidine perturb the electron density of the imidazole ring, further contributing to the transfer of charge from histidine to Fe2+. When NO binds to 5c heme, the d orbital electrons of the iron move to an unstable high spin state, with the formation of a transient 6c complex, that converts to 5c with the loss of the histidine-iron interaction [Dealing with metalloproteins like sGC poses significant challenges. Iron (Fe) is a transition metal and can exist in ferrous +2 and ferric +3 states. The Fe, and CO . The afferaction ,16.2/CO-ligated human HNOX domain in comparison with control apo-HNOX. A comparative modeling approach was used to estimate the structure of the human HNOX domain. The models were then further subjected to detailed dynamics investigation. Stability and hydrogen bond analysis revealed the binding pocket structural transitions in response to different ligands . Furthermore, essential dynamics (ED) were employed to identify noticeable structural variations. The following five complexes were examined; 1. Apo-HNOX (without gaseous ligands), 2. NO-HNOX (5c) 3. NO-HNOX (6c) with the bond between H105 and Fe removed to study the effects of bond-breaking (5c), 4. O2-HNOX (6c) 5. CO-HNOX (6c). Each system is examined for 50 ns.In this paper, we elucidate the atomistic dynamics of NO/O2. The human HNOX N-terminal domain (1–186 a.a) has 34% sequence identity with the nostoc bacterial template. Modeling results showed that 94.6% of the residues are found in favored regions of the Ramachandran plot and 97.8% in allowed regions. After energy minimization through YASARA  using models of differing sizes to balance speed and accuracy [2. Furthermore, the distance between the histidine imidazole ring and iron is high, in the NO-ligated system, in contrast to the CO and O2 ones. Moreover, O2- and NO-bonded complexes have bent conformations; however, the CO-ligated complex adopts a linear configuration with a 179° angle  was used in conjunction with Gaussian09 to parametrize the active site  and 124-129 (loop β) are significantly more mobile with a maximum RMSF of 4.2 Å for loop α. The 5c NO-HNOX system exhibited a similar behavior with a slightly smaller peak of 3.6 Å. In contrast, the highest peak of the CO-ligated system at 5.1 Å is observed at the loop β region. These observations suggest that the aforementioned sites possibly contribute to the release of heme from H105.Root mean square fluctuation (RMSF) stability analysis helps to investigate the mobility of residues under the influence of different ligands over the course of the entire simulation. The peaks represent areas of increased residual flexibility . The resg), provides a measure of the compactness of the protein by monitoring the effective size of protein. Rg is defined as the mass-weighted root mean square distance of a group of atoms from their common center of mass  is very similar to the O2-HNOX, except for the Y2 and R116 electrostatic interactions. The hydrogen bond fractional occurrence is high because the O2-HNOX (and apo-HNOX) system does not undergo as drastic conformational transitions as the NO/CO-bonded systems. Residues Y135, R137, R139, R116, and H105 are the major contributors of hydrogen bonds with the heme propionic acid moiety in all four systems. Weaker hydrogen bond interactions were also found for M1, Y49, A117, E138, and L142 in the different systems (2+) and H105 [2-HNOX and 6c NO-HNOX complexes , all systems were treated with the Ligplot tool [− and O2-ligated systems.To visualize the entirety of the electrostatic interactions of heme with the diatomic ligands  and MD simulations. MCPB.py generates two models of which the smaller one is used for bond and angle parameters calculation and the larger one for charge calculation. The hybrid functional method B3LYP [2/CO) while the force constant calculation was performed with the Seminario method [MCPB.py, a module of the Amber16 program package , which dod B3LYP  with theo method . This tho method . All calo method , with th2/NO/CO-HNOX and 5c NO-HNOX (where the Fe-His bond is broken) were explored with MD simulations. The calculated active site (H105-heme-NO/O2/CO) parameters were combined with the ff14SB force field to fully parametrize the protein systems [g) analysis was done to evaluate the systems’ stability throughout the simulation. Furthermore, hydrogen bond analysis and essential dynamics [Five different complexes, apo-HNOX, 6c O systems . The Amb systems . All sys systems  method w systems . All sysdynamics  were cardynamics . Visualidynamics , while tdynamics  and Gnupdynamics .The analysis of hydrogen bonds is indispensable to studying the binding modes of complexes. For our systems, the hydrogen bonds of heme with the distal binding pocket residues of the HNOX domain, Y2, Y135, S137, E138, L142, R139, and the proximal binding pocket residues H105, R116, P118 were analyzed with CPPTRAJ. Additionally, solute-solute, solute-solvent, and solute-solvent-solute bridges hydrogen bond analysis of the entire HNOX systems was also carried out. The lifetime of hydrogen bond occupancies is indicative of the overall binding mode occupancies throughout the simulation and was visualized with Gnuplot.2/NO/CO and apo-HNOX systems. Diagonalization of covariance matrices assists in calculating the principal components of motion [Essential dynamics were carried out to evaluate and interpret the multivariate data after detecting all correlated variable clusters. Furthermore, these correlated variables were converted to a small number of more meaningful uncorrelated variables . The CPPf motion . The traf motion .2 on the dynamics of the human HNOX domain, by employing a wide range of computational techniques. Residues in helix-f and loops α and β exhibited the most variable behavior, upon binding of the different gaseous ligands, highlighting the functional importance of these regions. These regions are located on the periphery of the inter-domain interface (HNOX-PAS) suggesting that they are involved in sGC activation. Among all complexes, 5c NO-HNOX experienced the highest positional displacement in helix-f and loop α, potentially facilitating the communication with adjacent sGC domains. The 5c NO-HNOX and 6c CO-HNOX residue R116 exhibits robust hydrogen bond interactions with the heme moiety, which could be critical to the communication with the neighboring domain. Interestingly, these interactions were either weak or absent in 6c NO-HNOX and 6c O2-HNOX. This work highlights important residues for sGC activation under physiological conditions that were not identified in our previous work [Soluble guanylate cyclase is a regulatory protein involved in a multitude of physiological processes in humans such as platelet aggregation, vasodilation, and neurotransmission. Deciphering the ways in which it is regulated and regulates other proteins requires detailed structural characterization. The HNOX domain is the starting point in the signal transduction mechanism of this heterodimeric, multidomain protein. In the current study, we analyzed the effect of the physiologically relevant diatomic gaseous ligands CO/NO/Oous work  on the b


KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown.kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding.Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.The online version of this article (10.1186/s12860-018-0177-9) contains supplementary material, which is available to authorized users. Cilia, organelles that project from the cell body and contain a microtubule-based axoneme, have numerous roles in development, signal transduction, and cell motility  and haveAlthough the ciliary membrane is continuous with the plasma membrane, the transition zone at the base of the cilium is believed to function as a ciliary gate involved in regulating protein entry and exit . While tkif17mw405 has diminished disc shedding. Additionally, cone expression of a constitutively active CaMKII leads to a two-fold increase in disc shedding in wild-type, but not kif17mw405 mutant fish. Taken together, this work supports a model in which phosphorylation of Kif17 regulates its accumulation in cilia. In cone photoreceptors, phosphorylated Kif17 in the OS participates in a cell-autonomous process to promote disc shedding.Functional roles of phosphorylation are often studied through specific mutagenesis of phosphorylation sites, which can be mutated to aspartic acid (D) or glutamic acid (E) to chemically imitate a constitutively phosphorylated residue or alanine (A) to imitate a constitutively un-phosphorylated residue –20. WhilPublished data suggest that the conserved NLS in the C-terminus of KIF17 can regulate the ciliary localization of KIF17 through a classical nuclear import mechanism . AlthougTo analyze nuclear localization, we calculated the ratio of average mCherry signal intensity between the nucleus and cytoplasm  promoter, also known as TaCP: Kif17-GFP, phospho-mimetic Kif17(S815D)-GFP, and phospho-deficient Kif17(S815A)-GFP . While previous work shows that Kif17-GFP can accumulate at the distal tip of the OS [While phosphorylation of KIF17 may regulate ciliary localization in mammalian cells, our main interest was in investigating the effect of KIF17 phosphorylation in ciliary photoreceptor OS in vivo. We generated comparable zebrafish Kif17 constructs specifically for cone photoreceptor expression with the cone-specific transducin-α gnat promoterKif17 in mature photoreceptors throughout the diurnal light:dark cycle could shed light on a potential function of KIF17. We performed qPCR on mouse retinas that were collected every four hours during a 24 h period beginning at light onset, indicated as Zeitgeber Time (ZT) 0, through dark onset (ZT 12), and stopping at ZT 20, or 20 h following light onset. Interestingly, there is a single peak in Kif17 expression in the mouse retina at ZT 4, just following light onset, which subsequently returns to a basal level , there is a second, more intense peak just following dark onset (ZT 16 and ZT 18)  on 5dpf zebrafish larvae that had been injected with the cone-expressing plasmids encoding either one of the three Kif17 transgenes or a soluble GFP control. Lacking transposase, the plasmids do not incorporate into the genome but rather facilitate episomal expression of transgenes. Interestingly, transient expression of the phospho-mimetic Kif17(S815D)-GFP was sufficient to drive an increase in the number of phagosomes compared to wild-type Kif17, phospho-deficient Kif17, or the soluble GFP control -GFP, and phospho-deficient Kif17(S815A)-GFP) on a mutant background called D-GFP, an fashion , 26. ThiTo measure disc shedding in the three transgenic lines, we collected 7dpf larvae at ZT 1.5 and performed TEM to analyze phagosomes Fig. a. There  analyzed in our experiments with the transgenic fish  as compared to controls -GFP promotes disc shedding, we sought to further investigate how the loss of D-GFP prokif17mw405 larvae as well as between the morning and evening peaks, we performed spline interpolation, a method of constructing a piecewise polynomial curve to smoothly fit the disc shedding data across all timepoints, for both the wild-type and kif17mw405 phagosome number data  phagosome number data  mice, while the evening peak was estimated to occur at about 1.7 h after dark onset for both genotypes  mice. After the evening peak, the number of phagosomes decreased at similar rates of 6 × 10− 3 and 7 × 10− 3 phagosomes/μm of RPE per hour in wild-type and Kif17tm1b(Bjc) mice respectively. Under all conditions, the half-life of a phagosome in mice is approximately 1–2 h. As in zebrafish, this analysis suggests little if any alteration in the kinetics of phagosome accumulation or digestion from loss of Kif17. In both mice and zebrafish, loss of Kif17 is associated with a decrease in the total number of phagosomes, supporting a role for KIF17 in promoting disc shedding in the mature photoreceptor.In addition to the deficiencies in disc shedding caused by loss of ice Fig. d withoutice Fig. e. In conIt has been suggested in an analysis using goldfish  that morkif17mw405 larvae  mice. While disc shedding is not completely ablated with loss of Kif17, we have previously proposed that additional factors, such as kinesin-II or diffusion, can compensate for defects in trafficking in Kif17 mutant photoreceptors [Kif17 mutants on disc shedding remains to be elucidated. Additionally, cone expression of transgenic phospho-mimetic Kif17 results in a cell-autonomous enhancement of disc shedding. CaMKII appears to be one of the kinases responsible for phosphorylation of Kif17 in photoreceptors in vivo, as cone expression of constitutively active tCaMKII promotes disc shedding in a kif17-dependent manner. However, additional investigation is required to identify other potential kinases or the phosphatase that may regulate removal of Kif17 from the OS. Ultimately, the phospho-mimetic Kif17 “gain of function” effects in promoting disc shedding combined with decreased disc shedding phenotype associated with genetic loss of Kif17 strongly implicate Kif17 in an important role regulating disc shedding at the level of the photoreceptor. Major advances have been made regarding the regulation of disc shedding, but most of that work has been at the level of phagocytosis by adjacent RPE cells [In this work, we find significant decreases in disc shedding in both PE cells –40. HowePE cells , and theKif17 mutant mice and zebrafish, diminished disc shedding is not accompanied by reduced size of the OS [Another significant conclusion from this work is that photoreceptors can exhibit considerable plasticity in the kinetics of ciliary OS turnover while still maintaining normal dimensions. It is well known that mouse rods shed approximately 10% of their length daily and maintain a steady-state length through compensating OS renewal . In the f the OS , suggestf the OS  and fluof the OS  pulse-laAs little is known about the photoreceptor-derived signaling that regulates disc shedding, the specific mechanism by which Kif17 promotes disc shedding is unclear. However, as PS exposure at the distal OS precedes disc shedding , it is pOur investigation of Kif17 loss of function required analysis of disc shedding at multiple timepoints because photoreceptor turnover is well-known to occur on a diurnal schedule , 29, 47,Overall, we show that phosphorylation of KIF17 enhances its localization along the length of the cone photoreceptor outer segment, where it promotes cell-autonomous disc shedding. As disc shedding has been predominantly studied within the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.Kif17tm1b(Bjc) mouse line (EUCOMM) as described previously [kif17mw405 zebrafish line as described previously [2 inhalation followed by cervical dislocation according to approved protocols.The animals used in these experiments were maintained in animal care facilities fully accredited by the American Association of Laboratory Animal Science, and used in accordance with the ARVO Resolution on the Use of Animals in Research, with applicable portions of the Animal Welfare Act and with the DHHS “Guide for the Care and Use of Laboratory Animals”. All experiments were approved and conducted in accordance with the Institutional Animal Care and Use Committee of the Medical College of Wisconsin. Mice were maintained in a pathogen-free barrier facility in cages of between 2 and 5 mice with access to dry food, water, and bedding material under 12 h:12 h light:dark cycle. We generated the A plasmid encoding CMV:KIF17-mCherry (GenBank MK046074) was generated as previously reported . tCaMKIIFor various experiments involving injected larvae, 9.2 nL of a working solution containing 25 ng/μL of a plasmid -GFP, TaCP:Kif17(S815A)-GFP, TaCP:tCaMKII-GFP, or TaCP:GFP), as well as 0.05% phenol red, were injected into ZDR embryos at the 1-cell stage. For stable line generation of TaCP:Kif17-GFP, TaCP:Kif17(S815D)-GFP, and TaCP:Kif17(S815A)-GFP, 10 ng/μL transposase RNA was included in the injection solution to facilitate genome integration [Commercial primary antibodies included: 6-11B-1, or mouse monoclonal anti-acetylated α-tubulin (Sigma), rabbit polyclonal anti-GNAT2 . The rabbit polyclonal anti-green and blue cone opsin antibodies were gifts from Dr. Thomas Vihtelic (University of Notre Dame) . The mou2. For ciliogenesis and transfection, cells were transferred to Opti-MEM (Gibco) at 60–80% confluency and transiently transfected with Lipofectamine 3000 (Invitrogen). Serum-starvation in Opti-MEM induced ciliogenesis and after 24 h, cells were fixed in 4% PFA for 20 min and subsequently immunostained with acetylated α-tubulin to indicate cilia and Hoechst to label nuclei. Because of the nature of transient transfection, there were a variety of transgenic expression levels. Approximately 20% of cells with a high level of expression in which transgene appeared to be highly vesicular or stuck in Golgi were excluded from analysis. For analysis, five separate transfections of each transgene in each cell line were performed. For ciliary localization in each cell line, the number of cells with mCherry labeling of the cilia was counted and divided by the total number of transfected cells. For nuclear localization, ImageJ was used to quantify the average intensity of both cytoplasmic and nuclear mCherry signal. Following subtraction of the average intensity of the background of each image (quantified from an area where there was no cellular fluorescence), the ratio of the cytoplasmic to nuclear average intensity was calculated.LLC-PK1 (ATCC CL-101), HEK-293 (ATCC CRL-1573), hTERT-RPE1 (ATCC CRL-4000), and IMCD3 (ATCC CRL-2123) were obtained from ATCC. Cell line authentication and testing of mycoplasma-negative were performed by ATCC. Additional testing of mycoplasma infection is performed biannually with MycoAlert (Lonza). LLC-PK1 cells were grown in Medium 199 (Gibco), HEK-293 cells were grown in DMEM (Gibco), and hTERT-RPE1 and IMCD3 cells were grown in DMEM/F12 (1:1) (Gibco). All media were supplemented with 10% FBS and 1% penicillin and streptomycin and cells were grown at 37 °C in 5% COEmbryos were injected with constructs as described above for mosaic expression and raised in phenylthiourea (PTU) to delay melanin pigment formation. At 4 dpf, larvae were anesthetized with tricaine and screened for GFP fluorescence. Immunofluorescence staining was performed on cryosections of 5 dpf GFP-positive larvae as previously described [kif17mw405 larvae were collected at nine different timepoints following light onset: 0 (light onset), 1.5 h, 3.5 h, 7.5 h, 10.5 h, 14 h (dark), 15.5 h, 17.5 h, 20.5 h. Collected larvae were processed as previously described for TEM [Kif17tm1b(Bjc) litter-mate mice were collected at eight different timepoints following light onset: 0 (light onset), 1.5 h, 4.0 h, 8.0 h, 12 h (dark), 13.5 h, 16 h, and 20 h. Mice were euthanized and whole eyes were processed as previously described for TEM [For transgenic or injected larva disc shedding analyses, zebrafish embryos were raised in phenylthiourea until 4 dpf when they could be screened for GFP fluorescence. GFP-positive embryos were then raised in regular fish water until either 5dpf  with 40 segments over the 24 h period (including the ZT 0 timepoint repeated at ZT 24) for increments of 0.615385 h. For mice, cubic spline interpolation was performed with 36 segments for increments of 0.6857143 h. For analysis, the maximum disc shedding rate during both the morning and evening was determined as the peak of disc shedding. To calculate the digestion rates of phagosomes, the maximum and minimum points of disc shedding for both the morning and evening shedding events was determined. Rates of phagosome digestion were subsequently calculated as the decreasing slope of the number of phagosomes over the period of between 25 and 75% of the total time between maximum and minimum. Phagosome half-life was calculated for both morning and evening as the time after the maximum peak until the phagosome number reached half-way between the maximum and minimum points.2O and stained with 1% aqueous uranyl acetate for 2 min. For each analysis, a total of five larvae were analyzed for each condition. For analysis, the number of both gold-labeled and unlabeled phagosomes was counted and divided by the total length of RPE measured.For immunogold analysis, 7dpf transgenic larvae were collected at 1.5 h after light onset or 14dpf wild-type larvae were collected at both 1.5 h after light onset and 1.5 h after dark onset. Larvae were fixed overnight in 0.1% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Larvae were washed in phosphate buffer, dehydrated with two 30 min washes of 70% ethanol, and incubated with a 2:1 mixture of LR white:70% ethanol for 1 h. Larvae were then incubated overnight in LR white at room temperature. In the morning, following two 30 min changes of LR white, larvae were embedded and polymerized at 55 °C for 48 h. Ultra-thin sections were mounted on 200 mesh formvar/carbon coated copper grids. Grids were incubated on drops of 0.15 mM glycine for 10 min, then on 5% BSA in 0.1 M phosphate buffer for 15 min. For labeling, grids were incubated on drops of antibody for 90 min. Grids were washed in 5% BSA in 0.1 M phosphate buffer and incubated with colloidal gold secondary (Electron Microscopy Sciences) for 1 h. Grids were washed in double-distilled HKif17 expression analysis, adult C57BL/6 mouse retinas were collected every four hours beginning at light onset over a 24 h period and placed RNAlater (Ambion) overnight at 4 °C. For zebrafish circadian kif17 expression analysis, 14dpf ZDR zebrafish eyes were collected at nine discrete timepoints following light onset: 0 (light onset), 1.5 h, 3.5 h, 7.5 h, 10.5 h, 14 h (dark), 15.5 h, 17.5 h, 20.5 h. For each biological replicate, three zebrafish eyes were pooled. For zebrafish opsin expression analysis, five whole 7dpf zebrafish larvae were pooled. Following sample collection, RNA was extracted, cDNA synthesized, and qPCR run as previously described [Kif17 primers were used [α primers were used as a reference gene [kif17 primers are depicted below.For mouse circadian ere used . For zebAll expression values were standardized to 1 for the light onset timepoint (ZT 0).Immunofluorescent images were acquired with a 100x, 1.30 NA oil-immersion lens (Nikon) on either an Eclipse TE300 (Nikon) microscope operating NIS-Elements (Nikon) and a Zyla sCMOS camera (Andor) or a C1 Plus-EX3 AOM Confocal System (Nikon) operating EZ-C1 (Nikon). TEM imaging was performed on a Hitachi H-600 with an ORCA-100 digital camera (Hamamatsu). Blinded image analysis was performed on de-identified, randomized images with ImageJ.Kif17 mRNA expression in either mouse or zebrafish retinas, a one-way ANOVA was performed. For qPCR analysis of opsin expression, a one-way ANOVA was performed for each opsin gene. For all experiments, following significance with either one-way or two-way ANOVA, a Bonferroni multiple-comparisons post-hoc test was performed to determine significance between groups. For all experiments, values are expressed as mean ± SEM and significance is labeled as *: p ≤ .05, **: p ≤ .01, ***: p ≤ .001, ****: p ≤ .0001. Non-significant values are unlabeled.For ciliary and nuclear localization of KIF17 in mammalian cell culture experiments, a one-way ANOVA was performed. Following significance, a Bonferroni multiple-comparisons post-hoc test was performed to determine significance between groups. For OS localization frequency and nuclear localization in zebrafish photoreceptors, a one-way ANOVA was performed. For line intensity analysis, a two-way ANOVA was performed. For single-timepoint disc shedding analysis in transgenic or injected larvae, a one-way ANOVA was performed. For immunogold phagosomes analysis in either transgenic or wild-type larvae, a two-way ANOVA was performed. For circadian disc shedding analysis in either zebrafish or mice, a two-way ANOVA was performed. For circadian Additional file 1:Figure S1. Phospho-mutations of S1029 regulate ciliary localization of KIF17. Figure S2. Phospho-mutations of S1029 mildly regulate nuclear localization of KIF17. Figure S3. Phospho-mutations of S815 regulate photoreceptor OS localization of Kif17. Figure S4. Mouse and zebrafish Kif17 are expressed rhythmically. Figure S5. Transient, episomal expression of phospho-mimetic Kif17(S815D) increases disc shedding. Figure S6. Opsin expression is largely unaffected by transgene expression. Figure S7. Rhodopsin immunogold labeling of phagosomes. Figure S8. Cone transducin-α immunogold labeling of phagosomes. Figure S9. Spline interpolation of zebrafish disc shedding data. Table S1. Spline interpolation of zebrafish disc shedding. Table S2. Spline interpolation of mouse disc shedding. (DOCX 5111 kb)Additional file 2:Plasmid map of CMV:KIF17-mCherry in GenBank file format (GB 17 kb)Additional file 3:Plasmid map of CMV:KIF17(S1029D)-mCherry in GenBank file format (GB 17 kb)Additional file 4:Plasmid map of CMV:KIF17(S1029A)-mCherry in GenBank file format (GB 17 kb)Additional file 5:Plasmid map of TaCP:Kif17-GFP in GenBank file format (GB 22 kb)Additional file 6:Plasmid map of TaCP:Kif17(S815D)-GFP in GenBank file format (GB 21 kb)Additional file 7:Plasmid map of TaCP:Kif17(S815A)-GFP in GenBank file format (GB 21 kb)Additional file 8:Plasmid map of TaCP:tCaMKII-GFP in GenBank file format (GB 19 kb)


Escherichia coli DnaB helicase•bacteriophage λ helicase loader (λP) complex. We show that five copies of λP bind at DnaB subunit interfaces and reconfigure the helicase into an open spiral conformation that is intermediate to previously observed closed ring and closed spiral forms; reconfiguration also produces openings large enough to admit ssDNA into the inner chamber. The helicase is also observed in a restrained inactive configuration that poises it to close on activating signal, and transition to the translocation state. Our findings provide insights into helicase opening, delivery to the origin and ssDNA entry, and closing in preparation for translocation.Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the  Chromosomal replicative helicases are hexameric protein ensembles that travel ahead of the advancing replisome, dissolving duplex DNA into templates for DNA synthesis. Melting of the duplex arises from ATP dependent translocation of the helicase along single stranded (ss) DNA, paired with inclusion/exclusion of DNA strands from an internal chamber. In bacteria, replicative helicases are closed protein rings, and the mechanisms associated with their loading onto chromosomal DNA, which is effectively an infinitely long polymer with no free termini, remain to be clarified. To assemble such entities on DNA, bacteria use specialized loading factors that mediate opening of the protein ring, guiding of ssDNA into the exposed chamber, and, finally, sealing of the helicase with ssDNA trapped inside . The arcE. coli: DnaA , c) theaA, RctB . UnwoundE. coli DnaC serves as the helicase loader and delivers the DnaB helicase to the bacterial origin; it is unrelated in sequence to λP, the loader that operates at the λ phage origin  by single particle cryoEM at 4.1 Å resolution. In the identified complex, we observe five λP loader molecules bound to the helicase at five consecutive DnaB subunit interfaces; the sixth DnaB interface has been breached, thus precluding a sixth λP from binding. We confirm the unanticipated B6P5 stoichiometry by native mass spectrometry. The λP loader restructures layers in the DnaB helicase comprised of carboxy-terminal (CTD) and amino-terminal (NTD) domains into novel right-handed open spiral configurations. Restructuring breaks one of the six helicase subunit interfaces to produce ~15 Å and ~20 Å openings in the CTD and NTD layers, respectively; these openings are of sufficient size to enable access by ssDNA to the internal chamber of the DnaB-helicase. Furthermore, reconfiguration forces the CTD layer of the helicase into a restrained inactive conformation wherein the ATP hydrolytic and DNA-binding properties are diminished, if not abolished. The restrained configuration of the CTD tier is poised to relax into the active conformation on expulsion of the loader from the complex. The NTD layer is also an important locus of conformational changes, which both contributes to closing of the helicase and prepares the helicase to interact with components of the replisome. Our findings reveal insights into mechanisms of opening and closing of the helicase, and provide a coherent structural view of helicase loading at the origin of DNA replication.Below, we address molecular mechanisms that accompany capture of the helicase by the loader in Stage II, and the transitions that link this stage to prior and ensuing events of the loading pathway. We report on the structure of the The structure of the BP complex was determined using cryo electron microscopy (EM) and tomography to a resolution of 4.1 Å . Five co6P5 stoichiometry predicted by the density map was verified using native mass spectrometry (MS)  and 419.7 kDa (B6P4) were observed at lower intensities. However, compositional heterogeneity was eliminated when ssDNA derived from the Oriλ phage replication origin was included. Native MS of the BP•origin ssDNA complex revealed a single entity with a mass of 459.5 kDa; this mass corresponds to the B6P5 complex bound to origin-derived 43-mer ssDNA (6P5.The unanticipated BE. coli DnaB bound to the DnaC helicase (BC) loader (The BP complex presents as a three-layered ensemble with approximate dimensions of 135 Å x 150 Å x 120 Å . Two of ) loader ; a more The third layer of the BP complex comprises the λP helicase loader . PresencIn the BP complex, five copies of the λP helicase loader bind to five consecutive DnaB subunit interfaces. Each λP protomer is comprised of a helical domain that binds in a deep gulley formed by an interface between two adjacent DnaB CTDs . The fiv2 interfaces described above each bury a substantial amount of accessible surface area . This feature is consistent with the observed high affinity between DnaB and λP B and λP . The finE. coli (Stage II/III) to those from A. aeolicus  and G. Stage IV ) are jusStage IV , Figure In comparison to the constricted closed planar ring form (PDB: 4NMN), the CTDs in the BP complex exhibit a right-handed pseudo-helical configuration characterized by an average helical rise per subunit value of ~4.1 Å, and a helical pitch of ~16 Å. Furthermore, we find that the CTDs exhibit an average helical twist of ~56.4° along the pseudo-helical axis. By contrast, the CTDs in the ssDNA bound form (Stage IV) exhibit an average helical rise per subunit of ~7.4 Å, a helical pitch of ~27 Å, and an average helical twist of ~60°.1•(CTD)2 substructure against all pairs of CTDs from the closed planar ring (constricted and dilated), and the closed spiral forms shows that the CTDs in the BP complex are inclined by ~20° towards the helical axis and the internal chamber, in comparison to those in the dilated closed ring form , and slightly larger than the constricted closed planar form , but much smaller than dilated closed planar form . Thus, the CTD tier in the BP complex adopts the constricted configuration.The λP helicase loader reconfigures the CTD layer in the BP complex into a conformation that differs substantially from previously described DnaB structures. Reconfiguration arises from three broad types of changes: (1) displacements of the CTDs along the pseudo-helical axis to form the open right-handed configuration; the average pitch of the resulting entity is ~40% shorter than the ensemble in the ssDNA bound complex, (2) changes to the helical twist of individual CTDs along the helical axis; the CTDs in the BP complex are underwound by ~3.6° with respect to those in the closed planar ring and closed spiral forms, and (3) inclination of the CTDs toward the helical axis; this change yields nucleotide-binding sites with configurations that are nearly optimal for catalysis (below). Collectively, these changes cause the rupture of one of the six CTD interfaces, between subunits A and B, and produce a ~ 15 Å gap between the two CTDs that span the breached interface; this gap is large enough to permit entry of ssDNA into the central chamber of the helicase. These findings have significant implications for opening and closing of the DnaB helicase during recruitment to origin DNA.G.st: R381, E382, G384; E. coli: R403, E404, G406) of each DnaB protomer contact two phosphate groups per subunit along the helical path of the CTDs ) and R442 (homolog of B.st K420) away from those seen in the translocating ssDNA complex  is similar to that seen in the closed constricted planar form, and differs markedly from the ~50 Å diameter of the corresponding structure in the ssDNA complex, which adopts the dilated conformation. Comparison of the helical parameters of the NTD tier from the BP complex  to those of the constricted closed ring form revealed an average helical rise per subunit of ~2.6 Å, a helical pitch of ~7 Å, and helical twist values per subunit that ranged from ~40° to ~70° along the pseudo-helical axis. Direct comparison between the open NTD spirals in the BP and ssDNA complex is complicated by their distinct configurations, constricted and dilated. Nevertheless, we find that the ssDNA complex shows similar pseudo-helical parameters: helical rise per subunit value of ~2.6 Å, a helical pitch of ~7.6 Å, and helical twist values per subunit that ranged from ~45 to 71° (comparison of PDB entries: 4ESV and 2R6A). Reconfiguration of the NTD layer in the BP complex into an open spiral creates a ~ 20 Å gap between the disrupted subunit interface. As with the opening in the CTD layer, the breach in the NTD layer is sufficiently large to provide access to the central chamber of DnaB to ssDNA.2). By contrast, the corresponding value for the BP complex is ~3100 Å2; this implies that the NTD and CTD layers are held considerably less tightly in the loader complex than in the closed ring.Direct remodeling of the CTD tier by the λP helicase not only allosterically remodels the NTD tier, but also changes the relationship between the NTD and CTD tiers in the complete DnaB helicase. In the closed planar structure, these two layers are essentially parallel. By contrast, the CTD and NTD layers of DnaB in the BP complex make an angle of ~15°; the corresponding value for the Stage IV form is ~7°. The change in the relationship between the two layers is also seen in the surface area buried. The NTD and CTD layers of the closed planar ring bury an extensive surface area  is known to be in equilibrium between two forms: dilated and constricted, with the dilated form representing the ground state; notably, nucleotide shifts the equilibrium towards the constricted state , one of which (tail-to-tail) will likely require more energy to open than the second (head-to-tail)  and trans (outer), these conformations describe distinct configurations of DnaB monomers. In the cis monomer, a helical hairpin substructure in the NTD points toward the CTD of the same chain, while in the trans monomer, this hairpin points away from the parent CTD , chain B (trans), chain C (cis), chain D (trans), chain E (cis), chain F (trans). However, in the ssDNA complex, we observe that the pattern of isomers differs: chain A (trans), chain B (cis), chain C (trans), chain D (cis), chain E (trans), chain F (cis). Thus, the mapping of CTDs between the two spiral forms of DnaB in closing Scheme I implies that a substantial rearrangement of the NTD tier must occur on helicase closing; this change consists of a cis to trans isomerization of DnaB subunits. In this scheme, closing is accompanied by disruption of each dimer interface in the NTD tier, a rotation of each isolated NTD, followed by re-establishment of the dimer, but with an adjacent, and distinct, DnaB subunit. However, the extensive interface found in the NTD tail to tail dimer  and ‘bottom’ (Chain A) . In addition, for our purposes here, ‘top’ and ‘bottom’ are inverted from that in . We noterent CTD . Cis and0 Å2 and ) is expecis-to-trans isomerization of DnaB monomers, and concomitantly the energetically costly disruption of NTD dimers, required of scheme I. A similar scheme to closing scheme II has been proposed for closing the helicase in the E. coli DnaB-DnaC complex  cells. Plasmids  were co-transformed into BL21(DE3). Standard methods were used to prepare starter cultures  media supplemented with 20 μg ml−1 kanamycin and 10 μg ml−1 streptomycin. Fermentation was allowed proceed at 37° C to an OD600 = 3 at a stir rate of 450 RPM and oxygen flow of 0.5 L min−1. At the three-hour point, protein expression was induced by bringing the culture to 0.5 mM isopropyl-thio-galactipyranoside (IPTG); induction was allowed to proceed for 5 hr at 37°C. This procedure typically yielded 200–300 g of cells. Harvested cells were resuspended in 50 mM Tris-HCl pH 7.6, 10% (w/v) sucrose, 500 mM NaCl, 2 mM DTT, 10 mM MgCl2 at a ratio of 5 ml per 1 g cells. The resuspended cells were flash frozen in liquid nitrogen and stored at −80°C until use.Bacterial expression of the isolated bacteriophage λP helicase loader under a variety of conditions yielded insoluble or poorly soluble material. As such, full length and truncated variants of the cultures , which wE. coli DnaB helicase • bacteriophage λP complex were lysed using a French press. After lysis, cell debris was removed by centrifugation. The BP complex in the resulting soluble fraction was precipitated by addition of 0.2 mg ml−1 ammonium sulfate and a 30 min incubation. The BP complex was resolved from both uncomplexed DnaB and λP using a combination of cation , affinity  and hydrophobic interaction  chromatography. The soluble isolated λP that emerged from the first step of chromatography was unstable and precipitated after coming off the column. All chromatography buffers used to prepare the BP complex contained 0.5 mM ATP. Purified BP complex , and hydrophobic interaction  chromatography. Purified DnaB•λPΔ102 was dialyzed into 20 mM Na-HEPES pH 7.5, 450 mM NaCl, 5% glycerol, 2 mM DTT, 0.5 mM MgCl2 and 0.1 mM ATP, concentrated with a Corning SpinX UF-20 100 kDa MWCO concentrator to ~4 mg ml−1, flash frozen in liquid nitrogen, and stored at −80°C until use.To prepare BP complexes with truncated λP (residues 103–233), pCDFDuet-λPΔ102-NHis and pET24-DnaB-EC were co-transformed into BL21 (DE3) cells and grown in LB media supplemented with 20 μg mlE. coli DnaB and the λP proteins confirmed the identity of both proteins (data not shown). All purification buffer components are listed in All protein chromatographic steps were performed using standard techniques . Unless A. aeolicus (AA) DnaB or A. aeolicus DnaC were expressed in E. coli Rosetta cells (Novagen) cultured at 37°C in Luria-Bertani (LB) broth supplemented with 50 μg ml−1 kanamycin and 34 μg ml−1 chloramphenicol.To prepare complexes for analyses, Cells that expressed AA DnaB were harvested and resuspended in 50 mM potassium phosphate pH 7.6, 500 mM KCl, 10% glycerol, 10 mM β-mercaptoethanol at a ratio of 5 ml per 1 g cells. Cells that expressed AA DnaC were harvested and resuspended in 50 mM HEPES pH 7.5, 50 mM potassium glutamate, 10 mM magnesium acetate, 5 mM β-mercaptoethanol at a ratio of 5 ml per 1 g cells. The resuspended cells were flash frozen in liquid nitrogen and stored at −80°C until use.E. coli in soluble form, instability of AA DnaC precluded efforts to work with the isolated protein, however, the AA BC complex could be readily purified. Inclusion of an excess of DnaC maximized the amount of the AA DnaB•DnaC (BC) complex in our preparation. Cells were lysed by sonication . The soluble fraction from this procedure was incubated at 65°C for 30 min; the AA BC complex remained in solution after removal of the precipitated material by centrifugation. The AA BC complex was further purified by a combination of affinity , cation exchange , and size exclusion . Purified AA BC complex was exchanged into 20 mM MES pH 6.0, 500 mM KCl, 10% glycerol, 1 mM dithiothreitol (DTT), 0.1 mM ethylenediaminetetraacetic acid (EDTA), flash frozen in liquid nitrogen, and stored at −80°C until use. Unless otherwise indicated all chromatographic steps were carried out at 4°C. All purification buffer components are listed in Cells in which AA DnaB and AA DnaC had been separately expressed were co-lysed at a ratio of 1 g DnaB: 2 g DnaC. The co-lysis approach was adopted because, although over-produced in 400 mesh carbon grids with holey carbon  were coated with ~50 nm of gold by evaporating 30 cm of 0.2 mm Au wire (EMS) onto 50 grids using an Edwards Auto306 evaporator and the carbon layer subsequently removed by plasma cleaning for 5 min in a Gatan Solarus plasma cleaner . Prior to sample adhesion, grids were plasma cleaned at 70% power, with a gas flow of 30%  for 60 s using a NanoClean model 1070 (Fischione Instruments).2, 0.5 mM ATP. 3.0 µL of the resulting solution was pipetted onto a fresh plasma cleaned grid; the sample was allowed to adsorb for 30 s at 100% humidity and 4°C, blotted for 3 s with a blot force of 4 and plunge frozen in liquid nitrogen-cooled liquid ethane. Sample adsorption and blotting were performed using a Vitrobot Mark IV . All grids were stored in liquid nitrogen until data acquisition.BP complex was freshly thawed on ice and diluted to a concentration of 1.5 µM in 20 mM Na-HEPES pH 7.5, 450 mM NaCl, 2 mM DTT, 0.5 mM MgCl-/Å2 s−1, an accumulated sample dose of 65 e-/Å2, and a defocus range of −1.0 to −3.0 μm. Three 24 hr sessions produced 2426 micrograph movies. .Grids were loaded into a Titan Krios , fitted with a Gatan K2 Summit  direct electron detector, operating at an acceleration voltage of 300 kV. Single particle movies were recorded at a pixel size of 1.07 Å with automatic hole targeting using LEGINON software suite . Images -/Å2 per tilt increment (subdivided over seven to nine frames) and a total accumulated sample dose of 90 e-/Å2. Data were collected with a pixel size of 1.76 Å and at defocus values of −2.8 μm, −6.1 μm, and −9.3 μm.In addition, five tilt series were collected from the same grids as used for the single particle collection. To minimize sample variation, tomography data were collected during a session that immediately following the single particle data collection described above. Tilted images were collected bi-directionally over a tilt range of −45° to +45° in 3° increments with a dose of 2.57 to 3.3 ehttp://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/). Additional details of our cryoET study are available here:  to calculate a homology model. To facilitate eventual refinement of our model against EM density maps, the homology model was constructed out of the highest resolution structures available for the DnaB NTD  and CTDB: 2R5U, ). Six iny MOLREP . In addiy MOLREP . As a rey MOLREP . Proteiny MOLREP  in whichy MOLREP .No atomic level information for the λP loader has been previously described. As such, an atomic model (~122/233 Cα) for the five λP protomers was constructed manually. The current resolution of our EM map did not allow the chain direction of λP to be determined, nor could amino acid side chains to be assigned to the structure. To address this limitation, we used CX-MS and protein-binding studies to determine that our EM maps included the carboxy-terminal domain of λP (Appendix and Inspection of our EM maps at lower contour (four sigma in PyMOL ) revealeModel building and visualization was performed in COOT .The BP model was refined using the real_space_refine routine in PHENIX . The refStructural analysis and visualization were carried out using the CCP4 software package , the UppA. aeolicus: 14–96; E. coli: 31–113; G.st: 14–96). The angle between the resulting planes from the NTD and CTD layers of DnaB was calculated as the arc-cosine of the quotient of the dot product and the cross product of the normal vectors of these planes.Analysis of the angular relationship between the NTD and CTD tiers of the various forms of DnaB was performed by fitting a plane to six points corresponding to the centers of gravity of the six CTD domains . The plA. aeolicus DnaB-DnaC) was carried out at a protein concentration of 5 µM. ssDNA  in our experiments were of two types: (1) a 43-mer sequence (5’ TGACGAATAATCTTTTCTTTTTTCTTTTGTAATAGTGTCTTTT 3’) derived from DNA unwinding element (DUE) of Oriλ , 200 V; ultrahigh vacuum pressure, 8–9 × 10−10 mbar; total number of scans, 100. The EMR instrument was mass calibrated using cesium iodide. The native MS spectra were visualized using the Thermo Xcalibur Qual Browser (version 3.0.63). Deconvolution was performed manually. For samples buffer-exchanged into native MS solution containing magnesium acetate, the deviations from expected mass ranged from 0.04% to 0.09% due to peak broadening from nonspecific magnesium adduction (see Native mass spectrometry (MS) of all samples  EcDnaB-λP-Oriλ derived ssDNA  was cross-linked with 2 mM disuccinimidyl suberate (DSS) overnight at 4°C; the crosslinking reaction was quenched with 50 mM ammonium bicarbonate. Isolated λP was not analyzed since it could not be produced in soluble form. We observed that crosslinking was more efficient when ssDNA was included in the crosslinking reaction. This factor, in combination with the finding that preparations of the BP•ssDNA complex contained a single entity . A further reviewer remains anonymous.Thank you for submitting your article "Mechanisms of Opening and Closing of the Bacterial Replicative Helicase" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers all found this manuscript interesting and the conclusions well justified. The contribution is captured by the following summary.E. coli replicative helicase, DnaB, in complex with the helicase loader protein from bacteriophage λ at an average resolution of ~4.1 A. Surprisingly, the structure shows that the DnaB hexamer binds 5 copies of the phage loader protein at its C-terminal side, with each λ P associating with two adjacent DnaB protomers. This unusual stoichiometry is supported by native mass spectrometry. In the BP complex structure, the DnaB hexamer adopts a constricted and spiral conformation that leads to a break in both the NTD and CTD tiers of the DnaB ring. This gap is wide enough to allow ssDNA to enter the central cavity and therefore supports a ring-breaking mechanism for loading of the E. coli replicative helicase. The authors further note that the DnaB ATPase ring is held in a restrained configuration that prevents the DNA binding loops and ATPase centers to adopt a conformation competent for ssDNA binding and ATP hydrolysis, respectively. However, this conformation likely is in some equilibrium with other states (see below for an issue that should be addressed in revisions). Overall these observations lead the authors to propose detailed models for both ring opening and closing of DnaB during replication initiation.The loading of replicative helicases onto DNA, often assisted by dedicated loader proteins, is an essential step during the initiation of DNA replication. In the manuscript "Mechanisms of Opening and Closing of the Bacterial Replicative Helicase", Chase et al., determine a cryo-EM structure of the E. coli loader protein, DnaC. The structure of the BP complex determined by Chase et al. thus represents the first near-atomic resolution structure of a DnaB-loader complex in the pre-loading state. Although the conformation of DnaB in the BP complex structure is very reminiscent of its configuration in the DnaBC complex, the new structure allows the authors to refine existing models for DnaB ring opening and closing and illustrates how two distinct loader proteins can remodel DnaB in a similar manner.Several crystal structures of DnaB have been determined previously, either in isolation or in complex with ssDNA, but structural information of DnaB-loading intermediates has been limited to a 25 A structure of DnaB in complex with its cognate Suggested revisions:1) The authors find that the inclusion of ssDNA improves the homogeneity of the complex when analyzed by native mass spectrometry and that it eliminates low abundant B6-P4 and B6-P6 assemblies. Moreover, it seems logical to assume that the use of a non-hydrolyzable nucleotide instead of ATP (which is hydrolyzed to ADP in the map) might stabilize the complex further. It is unclear why the authors did not attempt either approach, which may provide a higher resolution structure and would allow for more confident modeling of the λ P protein. Further explanation of the authors' reasoning would be appreciated.E. coli DnaB-DnaC complex. Although the authors acknowledge this in the main text, it would be useful to include this comparison in figure form as well, both for the ATPase domains and the NTDs.2) The conformation of DnaB in the BP complex seems very similar to its conformation in the 3) In Figure 2—figure supplement 1D, the authors show co-purification of untagged DnaB with the His-tagged C-terminal domain of λ P (aa 103-233) to support that the C-terminal region of λ P binds DnaB. However, this biochemical experiment lacks important controls, including a) a pull-down performed in the absence of His-tagged λ P protein to control for unspecific association of DnaB with beads and b) a pull-down using a tagged N-terminal fragment of λ P, which should not associate with DnaB according to the authors' interpretation. Moreover, the full-length λ P protein and the C-terminal fragment 103-233 both migrate around 20kDa in the gel despite their significantly different molecular weights. It would be helpful if the authors commented on this discrepancy.4) It is very difficult to identify individual particles in Figure 2—figure supplement 2D. The authors should consider showing a micrograph with better signal to noise and only highlight a few picked particles by circles. In addition, the 8 volumes that resulted from the 3D classification should be shown. There is also a discrepancy in the reported resolution of the cryoET reconstruction  in the legend and text (40 vs 25 A), which should be clarified.5) To demonstrate agreement of the built model with the EM map, the authors should provide the FSC comparing model with EM map.6) Figure 2—figure supplement 5: It is unclear what the authors mean by "heterogeneous" and "homogenous" refinement and, therefore, these approaches should be explained.7) Figure 6B: The authors' conclusion that the ATPase sites in the BP complex are misaligned is only weakly supported by the data shown. The authors report movements with distances much lower than the resolution of the structure. Although the authors acknowledge that the reported distances are only tentative, this practice is problematic, and we recommend that the authors should remove these distance measurements. In addition, it would be important to show densities for the side chains in the BP complex at the ATPase sites to demonstrate that the positioning of these side chains in the BP model is reliably supported by density in the map. Likewise, the authors should show densities for the bound nucleotide to support their conclusion that five ATPase sites are occupied by ADP instead of ATP.8) A reviewer was confused about the exact state of the Stage II complex. Because Stage III is the loader-origin-helicase complex, so, Stage II as described in this manuscript should be competent and ready to bind the origin DNA. But in several places, it was stated that the structure does not hydrolyze ATP (ATP binding sites are disrupted) and cannot bind ssDNA  (subsection “The DNA and ATP binding sites are disrupted in the DnaB• λP complex”). This seems to conflict with the mass spec data indicating the structure's ability to bind a 43-mer ori-lamda ssDNA , and the fact that five ADP molecules occupy the structure – meaning the structure has hydrolyzed ATP. This issue is alluded to in the summary- with a potential resolution? If the reported structure is indeed incompatible with DNA binding, does this mean the structure is a non-functional structure not found in a cell or perhaps a minor conformation in the cell?9) Related to the point in 8), the native mass spec shows the formation of DnaB-loader P-43-mer DNA . Did the authors attempt to visualize the structure, and if yes, is DNA visible, and is the structure distinct from the one reported here which is in the absence of DNA?10) Native mass spec revealed three species 6:5 (dominant), 6:4, and 6:6 stoichiometric binding. Did the authors identify the two non-dominant species (B6P4 and B6P6) in their 3D classification scheme of the cryo-EM dataset? Suggested revisions:1) The authors find that the inclusion of ssDNA improves the homogeneity of the complex when analyzed by native mass spectrometry and that it eliminates low abundant B6-P4 and B6-P6 assemblies. Moreover, it seems logical to assume that the use of a non-hydrolyzable nucleotide instead of ATP (which is hydrolyzed to ADP in the map) might stabilize the complex further. It is unclear why the authors did not attempt either approach, which may provide a higher resolution structure and would allow for more confident modeling of the λ P protein. Further explanation of the authors' reasoning would be appreciated.Both the BP and the BP-ssDNA complex samples were incubated in the presence of large excess of nucleotide (ATP). Immediately prior to taking the MS spectra, the samples were buffer exchanged into a volatile salt that does not include nucleotide. Nucleotide is excluded from this buffer because we found that this reduced the extent of non-specific adduction to the entities of interest and provided cleaner spectra. Inclusion of nucleotide during buffer exchange led to nonspecific binding and broadening of the MS peaks, which complicated their interpretation. From our native MS experiments, we infer that although nucleotide may play a role in formation of the BP complex, it does not appear necessary for stability of the BP complex. The finding that the BP complex is stable without nucleotide (both in the absence and presence of ssDNA) led us not to prioritize cryoEM experiments with non-hydrolysable ATP analogues. However, this will be something we pursue in the future.Aquifex aeolicus DnaB-DnaC helicase – helicase loader (BC) complex . The resulting mass estimates provide an important context for our efforts with the BP complex because the BC complex was treated in exactly the same way. In contrast to the BP complex, mass estimates for the BC complex reveal presence of five ATP molecules. From the comparison of the MS results of two samples, we conclude that the buffer exchange procedure differentially removes nucleotide molecules based on the individual complex’s affinities.In parallel, we performed native MS measurements with the distinct 2) The conformation of DnaB in the BP complex seems very similar to its conformation in the E. coli DnaB-DnaC complex. Although the authors acknowledge this in the main text, it would be useful to include this comparison in figure form as well, both for the ATPase domains and the NTDs.The published structural analysis of the DnaB-DnaC complex is a 25 Å negative stain EM analysis from the group of James Berger . Although fits of previously determined structures of DnaB and DnaC to the EM map are discussed in the published work, no atomic model of the DnaB-DnaC complex has been deposited in the structural database for EMDB entries 2321 and 2322. As such, there are no published coordinates for the DnaB-DnaC complex against which to compare our atomic model for the DnaB-LP complex.We have adjusted the narrative to reflect the above.3) In Figure 2—figure supplement 1D, the authors show co-purification of untagged DnaB with the His-tagged C-terminal domain of λ P (aa 103-233) to support that the C-terminal region of λ P binds DnaB. However, this biochemical experiment lacks important controls, including a) a pull-down performed in the absence of His-tagged λ P protein to control for unspecific association of DnaB with beads and b) a pull-down using a tagged N-terminal fragment of λ P, which should not associate with DnaB according to the authors' interpretation. Moreover, the full-length λ P protein and the C-terminal fragment 103-233 both migrate around 20kDa in the gel despite their significantly different molecular weights. It would be helpful if the authors commented on this discrepancy.To address the important missing control of whether the amino-terminal segment of λ P also binds to DnaB, we tested a construct that encompassed λ P residues 1 to 110  for co-expression and binding studies with DnaB. Multiple efforts to express this segment of λ P, in isolation or when co-expressed with DnaB, failed to show any expression of truncated λ P whatsoever. However, during co-expression, DnaB was clearly expressed; this observation suggests that our expression trials succeeded, but that the λ P construct of interest failed to express. As such, we conclude that this segment of λ P is unstable under the conditions tested. We have adjusted the text to reflect that this important control experiment could not be performed for technical reasons.We have corrected the labeling of the gels in Figure 2—figure supplement 1 to reflect the molecular masses for full-length and the C-terminal construct of λ P. This entailed replacing the original gel for panel C of Figure 2—figure supplement 1 with one that better depicted the molecular masses of DnaB and λ P. The corrected figure now shows that full length λ P migrates at ~27 kDa, whereas the histidine tagged C-terminus of λ P (residues 103-233) migrates around ~15 kDa.To address the question of non-specific binding of untagged DnaB•λP to NiNTA beads, we performed the experiment analyzed in 4) It is very difficult to identify individual particles in Figure 2—figure supplement 2D. The authors should consider showing a micrograph with better signal to noise and only highlight a few picked particles by circles. In addition, the 8 volumes that resulted from the 3D classification should be shown. There is also a discrepancy in the reported resolution of the cryoET reconstruction  in the legend and text (40 vs 25 A), which should be clarified.We agree that the micrograph shown in Figure 2—figure supplement 2 is difficult to identify individual particles. To remedy this issue, we have: (1) changed the displayed micrograph to one with higher contrast, and (2) elected to circle particles identified by template-based particle picking on the top half of the micrograph only, while leaving the bottom half of the micrograph unobstructed.To address the question of the 8 volumes from the 3D classification, we have created an additional figure . This figure depicts how we used the 3D classification procedure to clean our particle stack, as described in the Material and methods section and Appendix.On the question of the resolution of the cryoET volume; this is challenging to determine due to the relatively low number of particles used to generate the model . We estimate by visual comparison to filtered maps that the cryoET volume is in the 30-40 Å range and have edited the text in the manuscript to reflect this. Our use of cryoET to generate a low resolution model of the BP complex is described here . All instances of the reported resolution have been set to the same value.5) To demonstrate agreement of the built model with the EM map, the authors should provide the FSC comparing model with EM map.We have addressed this by using the Mtriage functionality within the Phenix software suite to compare our cryoEM map to our model. We have reported the data in a FSC plot in panel B of Figure 2—figure supplement 3. The resulting FSC comparing the model with our EM map results in two independently refined half-sets converging at 4.1 Å; this value is in agreement with the resolution of our map.6) Figure 2—figure supplement 5: It is unclear what the authors mean by "heterogeneous" and "homogenous" refinement and, therefore, these approaches should be explained.We have explained and defined these approaches more clearly in the Appendix of this manuscript . We have also altered panels A and B of Figure 2—figure supplement 5 to more clearly represent this procedure. Heterogenous refinement strategies involve the use of multiple ab initio models which each class is refined against, whereas homogenous refinement uses a single ab initio model for initialization of six classes.7) Figure 6B: The authors' conclusion that the ATPase sites in the BP complex are misaligned is only weakly supported by the data shown. The authors report movements with distances much lower than the resolution of the structure. Although the authors acknowledge that the reported distances are only tentative, this practice is problematic, and we recommend that the authors should remove these distance measurements. In addition, it would be important to show densities for the side chains in the BP complex at the ATPase sites to demonstrate that the positioning of these side chains in the BP model is reliably supported by density in the map. Likewise, the authors should show densities for the bound nucleotide to support their conclusion that five ATPase sites are occupied by ADP instead of ATP.We have removed the distance measurements but left the red arrows to reflect our best estimate for how the hairpin sub-structure has become mis-aligned at the level of the associated α carbon of residues known to be important for nucleotide hydrolysis (K440 and R442).We have added Figure 2—figure supplement 5 (panel B) that displays densities for the five nucleotide binding sites populated with ADP. These illustrations include insets of density that corresponds to the five observed ADP molecules.8) A reviewer was confused about the exact state of the Stage II complex. Because Stage III is the loader-origin-helicase complex, so, Stage II as described in this manuscript should be competent and ready to bind the origin DNA. But in several places, it was stated that the structure does not hydrolyze ATP (ATP binding sites are disrupted) and cannot bind ssDNA  (subsection “The DNA and ATP binding sites are disrupted in the DnaB• λP complex”). This seems to conflict with the mass spec data indicating the structure's ability to bind a 43-mer ori-lamda ssDNA , and the fact that five ADP molecules occupy the structure – meaning the structure has hydrolyzed ATP. This issue is alluded to in the summary- with a potential resolution? If the reported structure is indeed incompatible with DNA binding, does this mean the structure is a non-functional structure not found in a cell or perhaps a minor conformation in the cell?In the introduction, we have clarified that in Stages II and III the ATPase and ssDNA translocation activities are suppressed in the helicase – helicase loader complex, but that the complex retains the ability to bind ssDNA. As is noted above, our native MS data reveals that the BP complex binds to λ phage origin derived ssDNA, but not to other non-origin sequences of comparable length . However, the native MS data is silent on which entity in the complex is actually making contacts to ssDNA. DnaB is not known to show a preference for any ssDNA sequence, and, as such, it is striking that on binding the loader, the complex now exhibits a preference for origin derived ssDNA. From the appearance of the ssDNA binding site on DnaB, we infer that binding by DnaB may be severely disrupted, if not completely abolished. In the discussion, we have clarified that, taken together, our data imply that most, if not all, of the contacts to origin derived ssDNA may be made by the λP loader and that this finding sheds light on the nature of Stage III. As well, we propose that the structure captured by our EM analysis in indeed a physiological conformer, and that it might perform a previously uncharacterized activity: an origin specificity factor for the DnaB helicase.9) Related to the point in 8), the native mass spec shows the formation of DnaB-loader P-43-mer DNA . Did the authors attempt to visualize the structure, and if yes, is DNA visible, and is the structure distinct from the one reported here which is in the absence of DNA?Structural analysis of the BP-origin ssDNA complex is ongoing in the Jeruzalmi research group. At this time, our efforts are too preliminary for discussion.10) Native mass spec revealed three species 6:5 (dominant), 6:4, and 6:6 stoichiometric binding. Did the authors identify the two non-dominant species (B6P4 and B6P6) in their 3D classification scheme of the cryo-EM dataset?6P4 or B6P6 complexes suggested to be present in our preparation by our native MS experiments. In addition to the B6P5 volumes that we have discussed in the manuscript, we find evidence for a) incomplete DnaB rings or b) non-planar, cracked DnaB absent density corresponding to λ P. Our working explanation for this is that either these two populations (B6P4 or B6P6) are not stable enough to survive the plunge freezing process during grid preparation or that these populations were artifacts associated with the method of complex preparation. These efforts are detailed in the Appendix.We have exhaustively probed our cryoEM dataset for additional stoichiometries of the BP complex, and have not found evidence of the B


Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome seen in a predominantly elderly population, with multiple comorbidities including but not limited to: hypertension (systemic and pulmonary), chronic kidney disease (CKD), obesity, diabetes mellitus (DM) and coronary artery disease. Due to this intricate pathophysiology, definitive preclinical models are lacking and conventional HF therapeutic regimens fail in definitively treating HFpEF. This conundrum creates the need to classify individual patients into groups with certain cardiac and extracardiac abnormalities to successfully address HFpEF phenotypic diversity. To this end, Shah et al. . propose-/-Col4a3 mouse model of Alport nephropathy as a viable preclinical HFpEF model by recapitulating a range of HFpEF-related cardiac  and extracardiac abnormalities , summarized in Our group recently introduced the By taking advantage of the existing preclinical models, it is plausible to introduce other missing cardiac or extracardiac features in the Alport HFpEF model. Specifically, while we showed a lack of PH in Alport mice , the occWhile it has not been fully characterized, current evidence supports a long-standing theory that mitochondrial dysfunction is an important contributor towards both the normal aging process as well as age-related diseases . There i