The MCE domain of Mce4A has been crystallized as a domain-swapped dimer with the signature β-barrel fold. Solution studies show that the domains of Mce1A and Mce4A are predominantly monomeric and suggest that the helical domain is involved in lipid interactions.The mammalian cell entry genes ( Mycobacterium tuberculosis (Mtb), which is responsible for more than a million deaths annually, uses lipids as the source of carbon and energy for its survival in the latent phase of infection. Mtb cannot synthesize all of the lipid molecules required for its growth and pathogenicity. Therefore, it relies on transporters such as the mammalian cell entry (Mce) complexes to import lipids from the host across the cell wall. Despite their importance for the survival and pathogenicity of Mtb, information on the structural properties of these proteins is not yet available. Each of the four Mce complexes in Mtb (Mce1–4) comprises six substrate-binding proteins , each of which contains four conserved domains . Here, the properties of the various domains of Mtb Mce1A and Mce4A, which are involved in the import of mycolic/fatty acids and cholesterol, respectively, are reported. In the crystal structure of the MCE domain of Mce4A (MtMce4A39–140) a domain-swapped conformation is observed, whereas solution studies, including small-angle X-ray scattering (SAXS), indicate that all Mce1A and Mce4A domains are predominantly monomeric. Further, structural comparisons show interesting differences from the bacterial homologs MlaD, PqiB and LetB, which form homohexamers when assembled as functional transporter complexes. These data, and the fact that there are six SBPs in each Mtb mce operon, suggest that the MceA–F SBPs from Mce1–4 may form heterohexamers. Also, interestingly, the purification and SAXS analysis showed that the helical domains interact with the detergent micelle, suggesting that when assembled the helical domains of MceA–F may form a hydrophobic pore for lipid transport, as observed in EcPqiB. Overall, these data highlight the unique structural properties of the Mtb Mce SBPs. Approximately one quarter of the population of the world is latently infected with Mtb ] are important proteins that play a pivotal role in the import of lipid molecules and Mtb pathogenesis , when expressed in Escherichia coli, caused cell entry of E. coli into HeLa cells  and the substrate-binding proteins (SBPs) for the formation of the ABC transporter , which probably stabilize the Mce complexes , which is located elsewhere in the genome  and Acinetobacter baumannii (AbMlaD) have recently been characterized ]. A common feature of each of these proteins is that they all contain a conserved domain of approximately 100 residues, now referred to as the MCE domain, which is characterized by a seven-stranded β-barrel fold, although the sequence identity of these domains is very low. EcMlaD and AbMlaD have a single MCE domain, which forms a homohexamer in the assembled complex. EcPqiB and EcLetB have three and seven MCE domains, respectively, in a single polypeptide, which form stacks of homohexamers in the assembled complex. In contrast, each of the four Mtb mce operons encodes six different Mce SBPs and these SBPs have more domains compared with the E. coli and A. baumannii homologs. In this study, our main objectives have been to identify the various domains of MtMce1A and MtMce4A, guided by sequence analysis and secondary-structure prediction, and to perform a detailed structural characterization. The results of these studies show that the SBPs of Mtb have unique structural properties that differ from those of their bacterial counterparts.Although important functions of Mce proteins from 2.2.1.Mtb H37Rv was purchased from ATCC. Phusion DNA polymerase and the restriction enzymes used for cloning were purchased from Thermo Scientific  and New England Biolabs. The Ni–NTA chromatography resin was obtained from Qiagen .The genomic DNA of 2.2.Mtb H37Rv genomic DNA as the template with specific primers (Supplementary Tables S1 and S2). Each amplicon was cloned into pETM11 vector (EMBL) using a restriction-based cloning method, resulting in an N-terminal His6 tag followed by a TEV protease site, the MceA–F gene and a C-terminal His6 tag. For protein expression, the plasmid was transformed into E. coli BL21-RIPL competent cells. Overnight cultures were grown at 30°C until the OD600 reached 0.6, and expression of the protein was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16°C overnight. The cells were harvested by centrifugation at 4000g. The bacterial pellet was resupended in the desired lysis buffer with a suitable detergent (Supplementary Table S3). The cells were lysed by sonication and the lysate was centrifuged at 15 000g and 4°C for 30 min. The supernatant was then filtered  and the proteins were allowed to bind to the Ni2+–NTA matrix for 1 h. The beads were washed, and bound proteins were eluted from the Ni–NTA column using 400 mM imidazole in the elution buffer (Supplementary Table S3). At this step, the concentration of the detergent was reduced to 5 mM. The eluted protein was analyzed by 12% or 18% SDS–PAGE, concentrated  and injected onto a size-exclusion chromatograpy (SEC) column .Individual MtMce1A–1F and MtMce4A–4F genes were PCR-amplified using 2.3.36–148 and MtMce4A39–140), (ii) MCE+Helical+Tail domain (MtMce1A38–454 and MtMce4A36–400), (iii) Helical+Tail domain (MtMce1A126–454 and MtMce4A121–400), (iv) MCE+Helical domain (MtMce1A38–325 and MtMce4A39–320) and (v) Tail domain (MtMce4A321–400). The expression and purification protocols were similar to those used for the corresponding full-length proteins. Only the MCE-domain constructs (MtMce1A36–148 and MtMce4A39–140) are soluble in the absence of detergents, and different buffers were used for lysis and elution (Supplementary Table S3) when purifying these domains.Based on the secondary-structure analysis, MtMce1A and MtMce4A domain constructs were generated. They were cloned in pETM11 using restriction-free cloning methods: the constructs were named according to the secondary-structural features: (i) MCE domain , which was grown according to the protocol from Molecular Dimensions , which showed 100% incorporation of SeMet into the protein.For selenomethionine (SeMet)-labeled MtMce4A2.4.−1 was loaded onto a pre-equilibrated Superdex 200 10/300 column using an autosampler at a rate of 0.4 ml min−1 at 4°C in a Shimadzu HPLC/FPLC system. The samples were then passed through a refractive-index (RI) detector, a UV detector and subsequently through the MALS detector. The cumulative data collected from the UV, MALS and RI detectors were analyzed using the ASTRA software (Wyatt Technologies). The protein-conjugate analysis method was used to analyse the proteins that were complexed with detergent. The detergent was considered as a modifier and the recommended dn/dc value of 0.1473 ml g−1 for n-dodecyl β-d-maltoside (DDM) was used for the protein-conjugate analysis. Analysis of the soluble MtMce1A36–148 and MtMce4A39–140 constructs was performed without using the protein-conjugate protocol.SEC-MALS analysis of the purified MtMce1A and MtMce4A domains was carried out using a SEC column coupled to a miniDAWN TREOS light-scattering system (Wyatt Technologies). Purified protein at approximately 5–6 mg ml39–140 was subjected to buffer exchange  using a 10 kDa molecular-mass cutoff Amicon concentrator. MtMce4A39–140 was heated to 50°C in a thermocycler, with an initial 1 min incubation at 20°C followed by a 0.8°C increase per minute up to 50°C and a final incubation at 50°C for 1 min. The heated protein was then centrifuged at 10 000g for 5 min and the supernatant was injected onto a Superdex 200 10/300 column pre-equilibrated with a buffer consisting of 0.1 M MES, 0.7 M ammonium sulfate pH 6.0. The column was coupled to a MALS detector and was analyzed further to obtain the molecular mass.To understand the effect of heat and higher ionic strength on the oligomeric state of the MCE domain, purified MtMce4A2.5.−1. Secondary-structure calculations of the CD spectra of the MtMce1A and MtMce4A domains purified with DDM and without DDM were performed using the CDNN and BestSel software packages, respectively , the sample was heated from 22 to 92°C at a rate of 1°C min−1. The melting curves were calculated by comparing the spectra from 190 to 280 nm with the global fit analysis protocol as implemented in the Global3 software from Applied Photophysics.The MtMce1A and MtMce4A domains were diluted in water to obtain a lower buffer and salt concentration. The protein concentration used for CD measurements  was 0.05 mg ml2.6.36–148 and MtMce4A39–140 were buffer-exchanged into 20 mM ammonium acetate pH 6.8 using PD Miditrap G-25 columns . Mass spectra were measured on a 12 T Bruker solariX XR FT-ICR mass spectrometer using an Apollo-II electrospray ion source . The instrument was calibrated using sodium perfluoroheptanoic acid (NaPFHA) clusters and was operated with the FTMS Control 2.2 software. The mass spectra were further analyzed using the DataAnalysis 5.1 software.MtMce1A2.7.2.7.1.38–325, MtMce1A126–454, MtMce1A38–454, MtMce4A39–320, MtMce4A121–400 and MtMce4A36–400) at an initial concentration of 5 mg ml−1 as SEC can separate the protein–detergent complexes and the empty micelles  as a control. For each batch-mode concentration, 25 frames were collected.SAXS data for the purified Mce1A and Mce4A domains were collected on the B21 beamline at Diamond Light Source (DLS), UK. Data were collected based on the standard protocols for inline SEC-SAXS and batch-mode measurement using a PILATUS 2M two-dimensional detector at a sample-to-detector distance of 4.014 m and a wavelength of 0.99 Å. Inline SEC-SAXS measurements were collected for domains purified in the presence of the detergent DDM , versus q, where q is the modulus of the scattering vector. The scattering of the buffer was subtracted from the protein scattering using ScÅtter. The data were rebinned using in-house-developed software , forward scattering I(0) and maximum particle distance (Dmax) were calculated using PRIMUS. The molecular weight was calculated based on two methods: volume of correlation . Ab initio shape was generated using DAMMIN . The shell contains both DDM headgroups and solvating buffer, and the thickness of the shell was fixed at 10 Å. In practice, the aggregation number was kept fixed and the lengths of the long axis and of one of the short axes were optimized, whereas the length of the third axis was calculated from these two and the aggregation number. The Monte Carlo points were assigned an excess scattering length corresponding to the electron densities of C12 tails and heads for points in the core and in the shell, respectively, taking into account the glycerol content of the buffer. Similarly, the excess scattering length of the atoms of the protein was adjusted taking the glycerol into account. The scattering of a hydration layer was added to the protein in the places where it is not in contact with the micelle. The protein structure was divided into three domains, namely the MCE, helical and tail domains, to allow rigid-body refinement. The domains  were connected by soft restraints as described in Vilstrup et al.  in Phaser  of the MceA–F SBPs from Mtb Mce1–4 suggest that despite their very low sequence identity  they have a conserved domain architecture, such that each of them has four domains [Fig. 2a)]. The first domain is an N-terminal transmembrane (TM) domain (∼30–40 amino acids), which is predicted to form a single transmembrane helix, followed by a second domain with ∼100 amino acids mainly composed of β-strands , referred to as the MCE domain. The third domain is predicted to mainly consist of long helices (∼200 amino acids) and this domain is therefore referred to here as the helical domain. The fourth domain is predicted to be an unstructured domain and is referred to as the tail domain. Interestingly, the length of the tail domain varies between six and 260 amino acids between the various MtMceA–F SBPs, while the order and length of the other domains is well conserved. Additionally, the tail domains of MtMce1C, MtMce1D, MtMce4D and MtMce4F are proline-rich. Moreover, MtMce1E, MtMce2E, MtMce3E and MtMce4E contain a conserved sequence motif (referred to as the lipobox) in their N-terminus ]  in the presence of detergents. Given that all of these SBPs are predicted to have a similar domain architecture and secondary structure, further detailed domain-level characterization was performed for MtMce1A and MtMce4A. From secondary-structure predictions, the domain constructs of MtMce1A and MtMce4A categorized as MCE (MtMce1A36–148 and MtMce4A39–140), MCE+Helical (MtMce1A38–325 and MtMce4A39–320), Helical+Tail (MtMce1A126–454 and MtMce4A121–400) and MCE+Helical+Tail (MtMce1A38–454 and MtMce4A36–400) domains were successfully expressed in E. coli and screened to evaluate their solubility in the presence and absence of detergents.All six of the SBPs encoded in the Mce1 and Mce4 operons (MtMce1A–1F and MtMce4A–4F) were recombinantly expressed in 36–148 and MtMce4A39–140) were the only soluble constructs in the absence of detergents. The MCE+Helical+Tail and MCE+Helical as well as Helical+Tail constructs of MtMce1A and MtMce4A could only be purified in the presence of detergent, even though the transmembrane domain had been deleted in all of these constructs [Figs. 2b) and 2c)]. Additionally, extension of the soluble MCE constructs with one (MtMce4A39–154) or two (MtMce4A39–190) helical domains resulted in insolubility, indicating that the helical domain requires detergent for its solubility. This could be because under physiological conditions the helical domain is either embedded in the hydrophobic region of the cell wall or might be involved in interactions with the lipid substrates. The CD curves of the MtMce1A and MtMce4A domains [Figs. 2d) and 2e)] indicated mixtures of α-helical and β-sheet content for all of the MtMce1A and MtMce4A domains purified with DDM, whereas the soluble constructs (MtMce1A36–148 and MtMce4A39–140) showed a typical β-sheet-dominated spectrum (Supplementary Table S9).Interestingly, the MCE domains of both MtMce1A and MtMce4A , the structure of MtMce4A39–140 was determined using SeMet SAD phasing. The data-collection and data-processing statistics are reported in Table 1a)]. The electron-density map clearly defines the loops in the regions that define the swapping of the C-terminal part [Fig. 3c)]. The domain-swapped dimer is formed by the extension of residues 107–141 from one molecule into the other molecule. The swapped region contains two β-strands and an extended loop [Fig. 3a)].Structural studies were initiated on MtMce1Ab). The residues involved in formation of the seven-stranded β-barrel are Thr40–Ser46 (β1), Leu52–Met54 (β2a), Lys59–Gly65 (β2b), Ile65–Ser74 (β3), Arg81–Asp87 (β4), Thr99–Thr106 (β5), Ile107–Ile116 , His131–Val132 (β7a′) and Val137–Glu141 (β7b′). The residues from 107 to 141 are exchanged between the two monomers to complete the signature MCE fold. The overall structure has visible electron density for all of the residues corresponding to MtMce4A39–140 except for the N-terminal residues 1–31 and C-terminal residues 143–146. The latter residues correspond to residues encoded by the vector region.The secondary structure mainly consists of antiparallel β-strands, forming a β-barrel-like structure. The topology diagram for the swapped dimer is shown in Fig. 33.4.39–140 was surprising  studies were conducted. Interestingly, all of the purified MtMce1A and MtMce4A domains were predominantly monomeric in nature . The MtMce1A and MtMce4A domains purified in DDM showed two peaks in the elution profile corresponding to the protein–detergent complex (PDC) and empty detergent micelles, whereas MtMce1A36–148 and MtMce4A39–140, which are soluble and were purified without DDM, have a single scattering peak corresponding to the monomeric molecular mass . As the SEC-MALS analysis showed that both MtMce1A36–148 and MtMce4A39–140 are monomeric in solution and MtMce4A39–140 is a domain-swapped dimer in the crystal structure, the oligomeric states of MtMce1A36–148 and MtMce4A39–140 were also determined by native mass spectrometry (MS) at two different concentrations (5 and 50 µM). These studies further confirmed that both MtMce1A36–148 and MtMce4A39–140 are monomeric in solution at both concentrations .Given that the MCE domain of prokaryotes exists as a homohexamer in all of the recent studies, the domain-swapped dimer of MtMce4A39–140 calculated from the CD spectrum with the crystal structure showed a higher β-sheet content (39%) in the crystal than from the CD spectra in solution , indicating that the protein has more secondary structure in the crystallized condition. Moreover, thermal melting analysis of MtMce1A36–148 and MtMce4A39–140 showed that they undergo heat-induced conformational changes .Interestingly, comparison of the secondary-structure content of MtMce4A39–140 was exchanged into crystallization buffer and analyzed by SEC-MALS. Surprisingly, SEC-MALS analysis also showed only the presence of monomeric MtMce4A39–140 in the crystallization buffer . However, dimer formation was observed when MtMce4A39–140 was heated slowly to 50°C in the crystallization buffer . These observations suggest that incubation of this protein solution with the crystallization solution at 22°C probably facilitated the protein in attaining a different conformation, including the formation of a domain-swapped dimer. The dimer appears to be selectively crystallized, for example favored by better crystal contacts, compared with the monomer. There are other examples of full-length proteins and truncated domains which exist in different oligomeric states in solution but occur as domain-swapped dimers in the crystalline phase. These examples include barnase, cyanovirin-N, the N-terminal domain of Spo0A and the SH3 domain of Eps8, to name a few  are displayed as a function of the modulus, q, of the scattering vector. Structural parameters calculated from the scattering intensities are given in Supplementary Table S4. The radius of gyration (Rg) and maximum interatomic distances (Dmax) were determined to be 21.6 and 70 Å for MtMce1A36–148 and 21.7 and 80 Å for MtMce4A39–140, respectively . From SEC-MALS and SAXS, we know that the proteins exist as monomers in solution. Therefore, the ab initio shape of MtMce4A39–140 was fitted with two types of MtMce4A39–140 monomer: a compact monomer consisting of residues 32–106 from chain A and 107–145 from chain B of the crystal structure, and an elongated monomer consisting only of chain A as observed in the crystal structure. The missing N-terminal tag and linker sequences were modeled in these molecules using Robetta, as explained in Section 22 values of the compact and elongated models calculated against the experimental SAXS data were 10.0 and 2.0, respectively [Fig. 4b)]. Similarly, in the case of MtMce1A36–148, a template-based model (obtained from Robetta) was used to fit the SAXS data, and the χ2 values for the compact and elongated models were 14.0 and 11.0, respectively [Fig. 4a)], here also slightly favoring the elongated model. Further, the domain-swapped region of MtMce1A36–148 was optimized by rigid-body refinement and this improved the χ2 to 4.2. In summary, the elongated models fit relatively better than the compact model in both cases  and A. baumannii (AbMlaD) have been determined ]. Based on these homohexameric structures, two different mechanisms of lipid transport have been reported. The first is the Mla complex ferry transport mechanism, in which the Mla operon carries a single Mce gene (MlaD) with a single MCE domain. In this case, the lipids are shuttled between MalaFEDB and MlaA–OmpF by a shuttle protein  and A. baumannii MlaD (AbMlaD)  of 1.7 and 2.6 Å, respectively [Fig. 5a)]. The sequence identity between the MtMCE domain and the E. coli and A. baumanni MCE Mla domains is lower than 15% . The overall topology of the protein is conserved, with conformational differences mainly in the loop regions and a few other secondary-structural elements. For example, β2a (52–54) is only present in MtMce4A39–140 and not in EcMlaD and AbMlaD. The β4–β5 loop has an extra helix in MtMce4A39–140 and AbMlaD (a 45-residue insertion) and this helix is absent in EcMlaD. The β6–β7 loop in MtMce4A39–140 is a proline-rich loop, whereas it is lined with charged residues in EcMlaD and AbMlaD. In addition, density for the β6–β7 loop is missing in the EcMlaD crystal structure and is present in MtMce4A39–140 and AbMlaD. β7a and β7b are connected by a helix in MtMce4A39–140 and by a loop in EcMlaD, while β7a is absent in AbMlaD. The homologous β7b strand is much smaller in EcMlaD and AbMlaD compared with MtMce4A39–140.Superposition of the C39–140 was superposed with the MCE domains of EcPqiB1–3 and EcLetB1–7 monomers . The sequence identity between the MtMCE domains and the EcPqiB1–3 MCE domains ranges from 7% to 18% and that between the MtMCE domains and the EcLetB1–7 domains ranges from 13 to 26% . The superposition showed that the β-barrel fold is conserved and the observed differences are mainly in the loop regions. For example, β2a (52–54) is unique to MtMce4A39–140 and is absent throughout in EcPqiB1–3 and EcLetB1–7. The β3–β4 loop conformation present on the exterior surface varies amongst MtMce4A39–140, EcPqiB1–3 and EcLetB1–7. It is notable that the length of β3–β4 loop remains constant (four residues) in all of the MCE domains except EcPqiB3, which has 18 residues in the loop. Furthermore, the β6–β7 loop in MtMce4A39–140 has a different conformation compared with EcPqiB1–3 and EcLetB1–7. Amongst the available Mce SBP structures and MceA–F from MtMce1–4, MtMce4A39–140 has a maximum number of proline residues in the β6–β7 loop. The role of this proline-rich loop is not understood.Similarly, MtMce4A39–140, which contrasts with EcMlaD and AbMlaD. The PLL (β5–β6 loop) comprising the hydrophobic channel is much longer (16–27 residues) in EcPqiB1–2 and EcLetB1–7 when compared with MtMce4A39–140 (five residues). We found that the PLL in EcPqiB3 has only seven residues and it is the only MCE domain in EcPqiB and EcLetB which shares this feature with MtMce4A39–140. Interestingly, the conformation of the PLL varies throughout the MCE domains of EcPqiB1–3 and EcLetB1–7 . The central pore of all of the reported Mce SBP hexamers is comprised of highly hydrophobic residues, also known as the PLL, which allows the transport of small hydrophobic lipid molecules across the membranes. The variation in the length of the PLL depends on the transport mechanism followed by the particular Mce complex as well as the number of MCE domains that are present. For example, EcMlaD and AbMlaD have a smaller PLL (six residues) and they have a single MCE domain and follow a ferry-based transport mechanism. In comparison, the PLL is longer in EcPqiB and EcLetB (17–27 residues), which have three and seven MCE domains and follow a tunnel-based lipid-transport mechanism. It has been reported that the PLL of EcPqiB1–3 and EcLeTB1–7 follows the pattern φxxφφ, where φ denotes a hydrophobic amino acid and x represents any amino acid , it does not align with the other Mce SBPs from Mtb . Instead, the other Mtb Mce SBPs follow the pattern xxxφφ in the PLL, which aligns with EcMlaD and AbMlaD. Notably, the Mtb Mce SBPs and the MlaDs have only one MCE domain. Nevertheless, this conserved ‘duo’ of consecutive hydrophobic residues in the MtMce1A–F and MtMce4A–F SBPs indicate the formation of a hydrophobic pore. In addition, the helical domain of the MtMceA–F SBPs also has a high number of hydrophobic residues, although a clear ‘motif’ is not observed. The monomeric nature of MtMce4A39–140 is in contrast to the other Mce proteins  from E. coli and A. baumannii, which form a homohexamer  by superposing the MtMce4A39–140 monomer onto each of the six EcMlaD monomers. Interestingly, the protein–protein interface of the modeled homohexamer of MtMce4A39–140 has multiple steric clashes which will preclude the formation of homohexamers in MtMce4A [the clashes between chains A and B are shown in Fig. 5b)]. These clashes are absent in EcMlaD, AbMlaD, EcPqiB1–3 and EcLetB1–7, where homohexamers are formed. Overall, these comparisons show the different properties of MtMce4A39–140, although the core MCE fold is well conserved.The β5 strand and the hydrophobic β5–β6 loop  involved in forming the hydrophobic central pore have a different conformation in MtMce4A40 Fig. 5b by sup3.7.Supplementary Tables S5 and S6). The elution profile has two peaks: one for the PDCs and one for the empty micelles, showing that the PDCs and empty micelles are separated during SEC. The SAXS scattering data of the PDCs display a minimum at a scattering-vector modulus of 0.1 Å−1 followed by a broad bump. This suggests that the protein is interacting with the nearly intact detergent micelle. However, the scattering from the PDCs is distinctly different from that of empty micelles due to the additional strong scattering from the protein in the PDCs. This makes the forward scattering much greater for the PDCs . The empty micelle has a deeper minimum compared with the minima of PDCs around q = 0.1 Å−1. Additionally, the detergent is differently organized in the PDCs and therefore the shape of the secondary maximum also differs for PDCs and empty micelles  and 6b)]. In addition, a second variation of this model was generated by opening the helical domain to form an extended helix, keeping the tail domain far away from the MCE domain. This model is referred to as an ‘extended helical model’ [Figs. 7a) and 7b)].The crystallographic data for the et al., 2014et al., 2014From our experimental data, it is clear that the MCE domain is soluble and the presence of the helical domain requires detergent for purification. Therefore, the detergent micelle has to interact with the helical domain. Although the helical domain has a high number of hydrophobic amino acids, it also has polar residues which preclude the possibility of the helix being completely inserted into the micelle, as for a typical transmembrane protein. Furthermore, calculations of the SAXS intensity with the helix inserted into the core show that the SAXS intensity for these models smears out the minimum, so that it is not as deep as observed in the data. Therefore, a core-shell model of the detergent micelle was used where the core represents the hydrophobic tail (dodecyl chains) of the detergent molecules and the shell represents the head group (polar) and the water molecules associated with it , both the coiled-coil and extended models showed convincing fits, with χ2 values ranging from 3 to 20 .With these assumptions, both coiled-coil as well as extended helical models for each of the MtMce1A and MtMce4A constructs were optimized (ten independent runs) together with the micelle with an appropriate aggregation number to fit the SAXS data. For MtMce1A38–454  the extended helical model [Fig. 7a)] has a χ2 range of 15–24, compared with 22–50 for the coiled-coil model. The extended model fits the data well in the full q range, with a small deviation around the minimum, where the model curve is not quite low enough [Fig. 7c)]. The coiled-coil model is too small, with some deviations at low q, and the optimization compensates partly by displacing the MCE domain away from the Helical+Tail domain, leading to some disconnectivity in the structure [Fig. 6a)]. In the case of MtMce4A36–400 the data are not fitted well at high q values for both models, although the low-q data fit better to the extended model [Fig. 7d)]. Similar to the MtMce1A38–454 coiled-coil model [Fig. 6a)], the MCE domain and the Helical+Tail domain also become disconnected in the MtMce4A36–400 coiled-coil model [Fig. 6b)].In the case of MtMce1A121–400 extended and coiled-coil models have similar χ2 values in the range 4.6–8.0 . Both of these models have less deep minima with respect to the data. The MtMce1A126–454 extended model fitting has a χ2 range of 40–75, whereas the coiled-coil model fit shows a χ2 of between 114 and 182. Similar to the MtMce4A121–400 models, the minima are also less deep in the MtMce1A126–454 models . We have to accept that the tail domain is unstructured, with greater uncertainty in the structure prediction. This could also be a reason for the poorer SAXS fits for all of the constructs with the tail domain. The counting statistics of the data for the samples vary somewhat and therefore the χ2 values also vary, and it is observed that the χ2 values are often higher for data with good counting statistics. Therefore, we decided to also calculate R factors and weighted R factors, as used in crystallography. R factors are dominated by the high intensities at low q, whereas weighted R factors are a normalized measure of (χ2)0.5. The determined values are both in the range 1–5% (Supplementary Tables S5 and S6). They reveal that the deviation between data and fits is lower than the χ2 values suggest.The Helical+Tail domain fits for the MtMce4AThe above analysis confirms that the detergent micelle interacts with the helical domain irrespective of whether the helix turns back (as in a coiled-coil model) or is extended  in MtMce1A and MtMce4A. High-resolution information is needed to unambiguously conclude which of these two models is relevant under physiological conditions. Considering the low-resolution information in the SAXS data, as well as the possible errors in the generated MtMce1A and MtMce4A models, which are partly based on structural predictions, our analysis gives the best possible explanation for the observed SAXS data in a qualitative and in a semi-quantitative manner. The methods applied here for the analysis of MtMce1A and MtMce4A can be generalized for use for other membrane proteins as well as for membrane-associated proteins purified in the presence of detergents.4.Mtb Mce1A–1F and Mce4A–4F SBPs have been characterized. Each of the SBPs was individually expressed and purified from E. coli. Further, we have classified the Mtb Mce1A–1F and Mce4A–4F SBPs into four different domains based on secondary-structure prediction. The domain characterization shows the presence of a unique tail domain in the SBPs from Mtb that is not present in the other characterized homologs [Fig. 1a)]. The predicted length of the tail domain varies from 34 residues to 218 residues in the MtMce1A–1F and MtMce4A–4F SBPs. Further characterization shows that the full length as well as all of the domains of MtMce1A and MtMce4A remain as monomers in solution when purified individually. Only the MCE domain is soluble in the absence of detergents. The MCE domains of MtMce1A and MtMce4A occur as monomers in solution, as also shown by mass spectrometry. The crystal structure of the Mtb MCE domain reveals a β-barrel fold, as also found for its homologs, despite very low sequence identity (15% or less). The MCE+Helical and the Helical+Tail domain constructs require detergents for solubility. Further, SAXS analysis of MtMce1A, MtMce4A and their domains suggests that the helical domain may adopt the ‘coiled-coil’ or ‘extended helical’ conformation. In the coiled-coil model the MCE and tail domains are near each other, whereas the MCE and tail domains are far away from each other in the extended helical model. Irrespective of the conformation of the helical domain, it is very clear that the helical domain requires detergent for its stability and is either involved in interaction with the lipid substrates or embedded in the membrane. Structural analysis of MtMce4A39–140 suggests that the homohexamer cannot be formed, at least in Mce4A, due to multiple steric clashes. The fact that there are six Mce SBPs in Mtb suggests that the six MceA–F SBPs may interact with each other to form heterohexamers, where the helical domains of the six MceA–F molecules may form a channel as observed in EcPqiB piperazine-1-ethanesulfonic acid; IM, inner membrane; IPTG, isopropyl β-d-1-thiogalactopyranoside; MCE, mammalian cell entry; MES, 2-(N-morpholino)ethanesulfonic acid; MlaD, membrane lipid asymmetry; PDC, protein–detergent complex; PqiB, paraquat-inducible protein B; LetB, lipophilic envelope-spanning tunnel B; MS, mass spectrometry; Mtb, Mycobacterium tuberculosis; OM, outer membrane; SAD, single-wavelength anomalous dispersion; SBP, substrate-binding protein; SAXS, small-angle X-ray scattering; SEC, size-exclusion chromatography; SeMet, selenomethionine; Tb, tuberculosis; Tris, tris(hydroxymethyl)aminomethane.The abbreviations used are as follows. Ab, 39–140, native, 7ai3MtMce4APDB reference: SeMet-labelled, 7ai2PDB reference: 36–148, SASDJU9MtMce1ASASBDB reference: 38–454, SASDK32MtMce1ASASBDB reference: 126–454, SASDK22MtMce1ASASBDB reference: 38–325, SASDJZ9MtMce1ASASBDB reference: 39–140, SASDJV9MtMce4ASASBDB reference: 36–400, SASDJW9MtMce4ASASBDB reference: 121–400, SASDJX9MtMce4ASASBDB reference: 39–320, SASDJY9MtMce4ASASBDB reference: 10.1107/S2052252521006199/mf5054sup1.pdfSupplementary Results, Figures and Tables. DOI: https://www.sasbdb.org/project/1178/q9vh6j46wj/SASBDB data for the relevant Mce domains.:


Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity. Ycf1, a C-family member ATP Binding Cassette (ABC) transporter, transports glutathione and glutathione-metal complexes in yeast. Here the authors use cryo-EM and biochemical analysis to show how an intrinsically-disordered regulatory domain (R-domain) controls activity upon phosphorylation by engaging with a Nucleotide Binding Domain. These metals make up three of the top ten environmental toxins as defined by the CDC , marking Ycf1 as an attractive bioremediation target. Ycf1 also acts as a major redox sink and regulates redox levels in cytoplasm by sequestering glutathione after it is oxidized. In this way, Ycf1 serves a powerful protective function against metals and ROS, both of which are electrophiles that attack DNA, proteins, and the cell membrane.ATP-binding cassette (ABC) transporters regulate the movement of diverse molecules to support fundamental roles of the membrane including lipid homeostasis, ion transport, and detoxification. The Yeast Cadmium Factor 1 (Ycf1) performs such a protective function by transporting the tripeptide glutathione, the main non-protein thiol in cells, upon oxidation to maintain redox balance or after conjugation to toxic heavy metals such as cadmium, mercury, or lead into vacuoles6 including the medically important P-glycoprotein7, the Sulfonyl Urea Receptor (SUR18), and the Cystic Fibrosis Transmembrane conductance regulator (CFTR9). Rapid regulation is especially important to the C-subfamily of ABC transporters (ABCC family), which includes Ycf1 and CFTR, in particular because of their roles in regulating osmotic balance, neutralizing reactive oxygen species, or combating the effects of antifungal or anticancer agents in many organisms. Often, transporter phosphoregulation is mediated through phosphorylation on regions outside of the highly structurally conserved transport machinery. Instead, phosphorylation sites arise in long, disordered loops between domains or at the termini. In ABCC transporters, such regulatory elements are evolutionary additions to the canonical ABC exporter fold composed of two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs)  Fig. . These a11 and to lower open probability  and sensitivity to ATP concentration in the Ycf1 homolog CFTR15. Furthermore, mutations in 19 of the total ~130 residues of the CFTR R-domain are associated with mutants in the CFTR-linked lung disease, cystic fibrosis (www.cftr2.org). Phosphorylation can induce negative regulation as well, particularly on the Lasso motif in both Ycf116 and the Ycf1 functional homolog Multidrug Resistance Protein 1 (MRP117). Finally, the R-domain provides a surface for protein-protein interactions, including to other transporters such as SLC26A3 and the adapter protein 14-3-3β18.Disruption of this regulatory function can have profound cellular effects. Mutations of R-domain phosphorylation sites lead to loss of cadmium tolerance in Ycf119 and experimentally confirmed with circular dichroism20 and proteolysis analysis that estimate as low as 5% helical content20. However, more recent NMR experiments point to discrete helical fragments in the R-domain and direct interactions with NBD121. Meanwhile, cryo-EM structures of ABCC1, CFTR, and Ycf1 reveal a mostly disordered R-domain24 without assigned sequence for the phosphorylation sites, limiting our understanding of R-domain interactions. Structures of human CFTR and Ycf1 have revealed density for segments of the R-domain in proximity to NBD125. Unfortunately, the lack of a continuous R-domain sequence assignment in these models, especially in the phosphorylated regions, prohibits insights into the interfaces that govern R-domain regulation25.Despite recent advances in ABC transporter structural biology by cryo-electron microscopy (cryo-EM), the biophysical basis of these diverse R-domain interactions is poorly understood. Barriers to these characterizations hinge on the nature of the R-domain, which is predicted to be unstructured based on low sequence conservationSaccharomyces cerevisiae Ycf1 in two inward-facing conformations: wide (IFwide) and narrow (IFnarrow). We observe a resolved section of the R-domain that is most highly conserved in the ABCC family and includes the phosphorylation sites  and the NBD2 Walker B mutant E1435Q   is capable of efficient ATP hydrolysis. To enable structural studies of Ycf1, we expressed and purified two forms in the mg) Fig. , likely ain Fig. .5. These results are consistent with our ATPase data that show GSSG indeed stimulates ATPase activity at physiologically relevant concentrations (µM concentration range)  Fig. .25), with helical NMR assignments in CFTR21, and with evolutionary coupling analysis of co-evolving residues28 from a multisequence alignment of 13,723 homologs , interacts with the R-domain through an electrostatic or H-bond interaction from K617 to S903, another residue that is known to be phosphorylated  phosphorylated in three positions. The R-domain is helical (residues 920–929) in the C-terminal segment and lacks secondary structure (residues 901–919 and 930–935) in the other. The C-terminal helix was key to sequence assignment and aligns with partial models from CFTR cryo-EM structures . T911 also serves as part of a CKII kinase recognition motif (S/T-(x)n-S/T) that partially overlaps with the PKA-like kinase site  that were captured in our structures. To pinpoint which sites are most important for activity, we generated mutants of the phosphorylated residues  and important interacting residues (R206 and R716) and performed ATPase assays. The S908A mutant causes drastically lowered (~80% loss) ATPase activity and the T911A mutant causes ~50% loss in activity. Surprisingly, no change in ATPase activity in the S914A mutant was detected Fig. . This paNext, we investigated the interaction of these phosphorylated residue with their interacting partner residues (R716 and R206) Fig. . S908 anConversely, although S914A mutation did not impact ATPase activity, we investigated the impact of its interaction with R206 Fig. . We geneThe overall pattern of deleterious (S908A/T911A + R716A) and neutral or marginally positive (S914A + R206E) mutations aligns with the structural placement of the phosphorylated R-domain. We generated a double mutation of the R-domain (S908A/T911A) to investigate full attenuation of phosphorylation sites, but this resulted in highly destabilized protein that could not be purified. These results suggest that the observed interactions in our structure exhibit functional consequences when mutated and that partial compensation of single R-domain mutants by other phosphorylation sites can maintain some activity. The lack of stability of the double mutant suggests the complete loss of the R-domain-NBD1 interface.2 and ~1283 Å2 of surface area along the entire R-domain in IFnarrow and IFwide, respectively (~546 Å2 (IFnarrow) and ~425 Å2 (IFwide) specifically between NBD1 and the R-domain)  Fig. . In addirow Fig. .31. Analysis of the Ycf1 prediction shows a close recreation of the cryo-EM structures shown here, with a strikingly similar organization of the R-domain as the IFwide and IFnarrow structures. The R-domain region from 904-914 matches the ϕ−ψ backbone distribution and side-chain positions almost exactly, with several identical rotamer positions  contestThe AlphaFold2 analysis also recreates the intersection of the Lasso motif, R-domain, and the loop following TMD0 observed in our structures. Lower confidence regions show some divergence between the two structures, namely from 916–930, although both represent part of this region as α-helical. This region encompasses a clearly visible phenylalanine (F917) in the cryo-EM structure, but a movement of the downstream region of the α-helix, possibly due to a different overall conformation of the entire structure. Furthermore, the AlphaFold2 structure recreates clearly visible cryo-EM density for almost the entirety of the R-domain residues 883–897 and 849–860, excluding ~20 residues; however, the position of these residues cannot be built with confidence and was excluded from the final model ain Fig. . Thus, oThe observed R-domain–NBD1–lasso contacts are consistent between IFwide and IFnarrow with minimal changes between these domains Fig. . Instead30, are brought into contact upon transition to IFnarrow  suitable for ATP binding and initiation of transport. We propose this yields a primed, active state ready for transport.In contrast to the interaction sites themselves, the major structural consequences of the IFwide to IFnarrow transition are induced at the ATP-binding sites. These sites not only move closer in IFnarrow but also rotate to occlude the non-functional ATP-binding site in NBD1 Fig. . Notablyrow Fig. . Overallrow Fig. 22,32. Th27. The presence of an ordered R-domain position poses a surprise, since many alternate models exist for the R-domain mechanism that include a completely disordered R-domain or an R-domain that resides only between NBD1 and NBD2 in a semi-ordered state.The structures of Ycf1 determined here reveal previously unobserved interactions in the ABCC family of ABC transporters that inform on how they are regulated. This includes an inward-open conformation with an intact and endogenously phosphorylated R-domain tightly bound to NBD1. Our structures provide a more complete model than previous ABCC structures, which resolve small fragments of the R-domain, do not resolve the phosphorylation sites, and/or do not assign the sequence for this region11 and the requirement for an intact N-terminus containing the Lasso motif of CFTR for activity33. The extensive R-domain and NBD1 interactions increase the ATPase rate in a manner consistent with CFTR, where R-domain phosphorylation potentiates ATP binding and hydrolysis but otherwise does not induce transport activity alone34. Our structures also are consistent with the visible R-domain density in CFTR and polyalanine models built into this density . The most pronounced difference is an R-domain register shift of 40 positions (901-927 in this structure compared to 860-874 in 7MPE). Outside of the R-domain, both structures match closely with a Cα RMSD of 2.3 Å and similar overall topology. It is unclear whether this is another state of the R-domain but several pieces of evidence support its placement in the model presented here. First, continuous density was visible from the end of the R-domain to the 2nd TMD that allowed for model building of our structure. This assignment allowed for placement of key residues including F917 in clear density in a hydrophobic pocket and ultimately placement of phosphorylated residues of the R-domain that closely match thos in CFTR. Second, our mutagenesis data provide consistent evidence of the functional importance of R-domain interfaces when mutated on either the R-domain or its corresponding binding surface  compared to 7MPE and the AlphaFold2 model (6.3 Å). An even higher discrepancy exists between the alignments of just the NBD1 + R-domain region (610–927) performed to account for domain motions between structures (IFwide to AlphaFold2 model: 2.6 Å versus 7MPE to AlphaFold2 model: 10.7 Å). High predicted pLDDT scores (a measure of confidence in AlphaFold2) along the conserved phosphorylation motifs (>70 at D909) confirm the placement of the R-domain and suggest high sequence co-evolution in this region, particularly between positive charges on NBD1 and the phosphorylatable R-domain residues. The AlphaFold2 model also shows reasonable placement of the remaining R-domain segments, including those preceding the start of R-domain model (880–900) and part of the R-domain after NBD1 (849-859) consistent with visible electron potential density from our cryo-EM maps. Although these segments are still too disordered to reliably be modeled without another source of distance restraints, they are more consistent with the placement of the entire R-domain in our models.36. This suggests that perhaps a common mechanism exists with the R-domain as well, although further biophysical and computational studies will be needed to uncover how.It is unclear why there are such substantial differences in the placement of the R-domain. The alternate arrangement of the R-domain in the 7MPE model is physically plausible as it places a cluster of electronegative side chains where our model has placed phosphates. Because of the remaining “slack” in the R-domain, it is possible that both models are correct and represent different substates of the bound R-domain structure. Recent reports from CFTR suggest that the R-insertion can alter its secondary structure and register along NBD137. The high convergence of several important effects near one region where the R-domain, NBD1, R-insertion, and Lasso motif come together suggest the general importance of the R-domain interactions observed in our structures.Our results also rationalize key clinically relevant findings related to other ABCC family members. The extensive electrostatic, hydrogen bonding, and hydrophobic interactions on the R-domain explain how mutations distal from phosphorylation sites may nevertheless contribute to dysfunction. Additionally, the site equivalent to the most widespread cystic fibrosis linked mutant, (CFTR delF506) Ycf1 F713, is located in NBD1 directly adjacent to the phosphorylation sites of the R-domain and is coordinated by cation-pi interactions with two conserved arginine side chains, R765 and R1150  of an important class of transporters, the ABCC family. The presence of a single defined binding site for the conserved part of the R-domain confirms an anticipated but elusive binding site and may provide implications for several other types of transporters where such long phosphorylated unstructured domains or loops are widespread. Such a defined binding site could also provide a basis for allosteric modulation of transporter function in a diverse array of settings.S. cerevisiae YCF1 (Yeast Cadmium Factor 1) gene was codon-optimized and cloned into the p423_GAL1 yeast expression vector as an N-terminal Flag (DYKDDDDK) and C-terminal decahistidine (10X His) tagged fusion protein (GenScript)  was transformed with the Ycf1 expression construct and a 50 mL primary culture grown for at least 24 h at 30 °C with shaking at 200 rpm in SC-His media . A secondary 750 mL culture of SC-His media was inoculated with 2% of the primary culture (15 mL) and grown under the same growth conditions for an additional 24 h prior to induction by adding YPG media  from a 4X YPG media stock. The culture was grown for an additional 16 h at 30 °C prior to harvesting by centrifugation at 5000 × g for 30 min at 4 °C.For protein expression, the g for 1.5 h prior to storage at −80 °C. Membranes were solubilized in resuspension buffer /0.05% cholesteryl hemisuccinate (CHS) supplemented with protease inhibitor as described above) at a ratio of 15 mL/g of membrane at 4 °C for 4 h. Solubilized membranes were clarified by centrifugation at 34,155 × g for 30 min at 4 °C. The clarified supernatant was filtered through a 0.4 µM filter to remove the insoluble fraction and supplemented with 30 mM Imidazole pH 7.0 immediately before loading at a flow rate of 2 mL/min onto a 5 mL Ni-NTA immobilized metal affinity chromatography (IMAC) column (Bio-Rad) equilibrated in Buffer A . Following loading, the column was washed with 10 column volumes (CV) of Buffer A to remove nonspecifically bound proteins then followed by a gradient of Buffer B  consisting of the following step sizes: 6% (10CV), 10% (2CV), 16% (2CV), and 24% (2CV). Protein was eluted with 4CV of 60% buffer B and immediately diluted 10-fold with Buffer A prior to concentration and 3 rounds of buffer exchange to remove excess imidazole by centrifugation at 3095xg at 4 °C in 100 kDa cutoff concentrators (Amicon). Concentrated, buffer exchanged sample was lastly purified by size exclusion chromatography (SEC) at 4 °C by injecting sample onto a Superose 6 Increase 10/300 GL column  equilibrated in SEC buffer  supplemented with either 0.01% LMNG/0.001%CHS or 0.06% digitonin and immediately used for biochemical assay or cryo-EM grid preparation following quantification by BCA Assay (Pierce).For protein purification, harvested cells were resuspended with ice-cold lysis buffer ) at a ratio of 3.2 mL/g of cell pellet. Resuspended cells were lysed on ice by bead beating with 0.5 mm glass beads for 8 cycles consisting of 45 seconds of beating, with 5 min between cycles. Lysates were collected by vacuum filtration through a coffee filter and membranes harvested by ultracentrifugation at 112,967 × 2 . Movies of E1435Q Ycf1 contained 60 frames with a per frame exposure of 0.9 electrons /Å2 dose rate .Cryo-EM grids for WT and E1435Q Ycf1 were similarly prepared. Immediately following SEC purification, 5 µL of concentrated WT (15.2 mg/mL) or E1435Q (5.94 mg/mL) Ycf1 sample was applied to a CFlat-1.2/1.3-4C-T (WT Ycf1) or QF-1.2/1.3-4Au grid (E1435Q Ycf1) purchased from Electron Microscopy Sciences. Grids were placed inside of a Leica EM GP2 equilibrated to 10 °C and 80% humidity. Following a 10 s incubation, the side of the grid to which sample was applied was blotted on Whatman 1 paper , then immediately plunge frozen in liquid ethane equilibrated to −185 °C. A total of 3,262 movies were captured for WT Ycf1 using Serial EM software on a Titan Krios at 300 kV equipped with a K2 Summit detector (Gatan) at Arizona State University. For E1435Q, 8,499 movies were captured on a Titan Krios at 300 kV equipped with a K3 Summit detector (Gatan) at the Pacific Northwest Center for Cryo-EM. Movies of both WT and E1435Q Ycf1 samples were collected at 22,500X magnification with automated super resolution mode and defocus ranges of −0.5 to 2.8 µm (WT Ycf1) and −0.9 to −2.1 µm (E1435Q Ycf1). Movie frames for WT Ycf1 contained 40 frames with a per frame exposure of 1.4 electrons /Å39 and 3.140) and cisTEM41. Drift correction was performed using MotionCor242 to generate an image stack with a pixel size of 1.031 Å/pixel. The contrast transfer function (CTF) was estimated for dose-weighted micrographs using CTFFIND4.1 prior to particle picking using a reconstruction of WT Ycf1  in which NBD2 was poorly resolved. Maps from RELION were further refined in cisTEM and used for manual model building. ResMap was used for local resolution estimation performed on cisTEM maps45. A summary of the data processing workflow and final EM map quality is reported in Supplementary Figs. The Ycf1 E1435Q dataset was processed in RELION . Nonlinear regression analysis of data fit with the Michaelis-Menten equation in GraphPad Prism 9 was used to generate kinetic parameters for at least three technical replicates.For evaluating ATPase activity, WT and E1435Q Ycf1 were expressed and purified as described above in buffer containing 0.01% LMNG and 0.001% CHS. ATPase rates were determined at 30 °C using an enzyme-coupled assay63. Data were fit using nonlinear regression in GraphPad Prism 9 to derive EC50 and Vmax values.Substrate-stimulated ATPase activity was measured as described above and in the presence of varying concentrations of oxidized glutathione (GSSG) Fig. . The conYcf1 phosphorylation was assayed by in-gel phosphoprotein staining. Dephosphorylated Ycf1 for these experiments was generated by treating SEC-purified Ycf1 (10 µg) with Lambda phosphatase  for 1 h at 30 °C in 25 µL reaction volume. Following phosphatase treatment, dephosphorylated Ycf1 was subject to a second round of SEC purification to remove excess enzyme immediately prior to use for subsequent biochemical analysis. For in-gel phosphoprotein staining, equal quantities (5 µg) of untreated Ycf1 and lambda PP (NEB) treated samples were separated on two separate 10% SDS- PAGE gels, one of which was then stained with Pro-Q Diamond Phosphoprotein Gel Stain (Thermo Scientific) following the manufacture’s protocol to detect protein phosphorylation. The second gel was simultaneously stained with Coomassie Brilliant Blue R-250 as a control for monitoring total protein levels. Full size gels are provided in Source data file.To measure quantitative phosphorylation of purified Ycf1, 10 µg of Ycf1 were Trypsin/LysC  digested with an S-Trap column  using the manufacturer’s suggested protocol following reduction with DTT and alkylation with IAA with ProteaseMax (0.1% Promega) added to the digestions. Altogether, 2500 ng of protein were loaded for each run. The LC–MS/MS analysis was performed using a Q-Exactive Plus  mass spectrometry with an EASY-Spray nanoESI. Peptides were separated using an Acclaim Pepmap 100 trap column (75 micron ID × 2 cm from Themo Scientific) and eluted onto an Acclaim PepMap RSKC analytical column  with a gradient of solvent A (water and 0.1% formic acid) and solvent B (acetonitrile and 0.1% formic acid). The gradients were applied starting with 3–35% Solvent B over 90 min, then 25–50% solvent B over 20 min, 50–95% solvent B over 5 min, and a 100% solvent B for 10 min, and then 3% solvent B for 10 min.64. A survey scan at 70,000 resolution scanning mass/charge (m/z) of 350–1600 was performed with an automatic gain control target of 1e6 with a maximum injection time (IT) of 65 msec, then a high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS) at 37 NCE  of the 11 highest intensity ions at 17,5000 resolution, 1.5m/z isolation width, 5e4 AGC, and 65msec maximum IT. Dynamic exclusion was used to select an m/z exclusion list for 30 s after single MS/MS and ions with a charge state of +1, 7, >7, unassigned, and isotopes were excluded.Data were collected with a flow rate of 300 nL/min applied with a Dionex Ultimate 3000 RSLCnano system (Thermo Scientific) with data dependent scanning using Xcalibur v 4.0.27.19S. cerevisiae protein database (https://www.uniprot.org/proteomes/UP000002311), and a database of common contaminant proteins  with Thermo Proteome Discoverer v 2.4.0.305 (Thermo Fisher Scientific). Fully tryptic peptides with up to two missed cleavage sites were considered in MS/MS spectral matches. The variable modifications considered included methionine oxidation (15.995 Da), cysteine carbamidomethylation (57.021 Da), and phosphorylation (79.966 Da) on serine, tyrosine, and threonine. XCorr score cutoffs at 95% confidence were used to identify proteins using a reverse database search65. Identification results from proteins and peptides were further analyzed with Scaffold Q + S v 4.11.1 , which integrates various search results  and using Bayesian statistics to identify spectra66. We considered protein identification that satisfied the criteria of a minimum of two peptides with 95% confidence levels for protein and peptide.Search of MS and MS/MS data were performed against the Uniprot Fixed amounts of Lambda PP treated and untreated Ycf1 protein (6 µg) sample were incubated for 1 h on ice with vary amounts of 100 mg/mL trypsin from bovine pancreas (Sigma) at varying final concentrations . Samples were brought to 20 mL total volume with a buffer containing 20 mM Tris, 300 mM NaCl, and 0.05%LMNG. The reaction was stopped by adding 1 µg/mL of soybean trypsin inhibitor (Sigma) and incubating for an additional 15 min on ice. Next, 5 µL of each reaction were mixed with 1X SDS loading dye and 100 mM DTT from each reaction and separated on a 10% SDS- PAGE gel before visualizing with Coomassie Brilliant Blue R-250 staining.Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


In this paper, significant attention is paid to the retention of phenolics in extrudates and their health effects. Due to the large number of recent articles devoted to total phenolic content (TPC) of input mixtures and extrudates, the technological changes are only presented for basic raw materials and the originating extrudates, and only the composites identified has having the highest amounts of TPC are referred to. The paper is also devoted to the changes in individual phenolics during extrusion . These changes are related to the choice or raw materials, the configuration of the extruder, and the setting the technological parameters. The results found in this study, presented in the form of tables, also indicate whether a single-screw or twin-screw extruder was used for the experiments. To design an extrusion process, other physico-chemical changes in the input material must also be taken into account, such as gelatinization of starch; denaturation of protein and formation of starch, lipids, and protein complexes; formation of soluble dietary fiber; destruction of antinutritional factors and contaminating microorganisms; and lipid oxidation reduction. The chemical changes also include starch depolymerization, the Maillard reaction, and decomposition of vitamins. As a thermomechanical process, extrusion cooking is used for the production of a large number of food products, e.g., breakfast cereals , confectThe extrusion process results in numerous transformations, such as gelatinization of starch, denaturation of proteins , formatiChenopodium, dried mango, tigernut flour, dried green banana flour, and millet have also been tested (see the following text and tables), often with additives, which are usually materials rich in phenolics and/or waste from the food industry.The main ingredient of extrudates is usually cereal flour . Oat, chickpea, soybean, white ginseng root hair, cassava flour, germinated The two basic types of extruders are single- and twin-screw mechanisms, in which the screws co- or counter rotate. The temperature and pressure in the extruder are not only influenced by the shape of the screw, the technological parameters (mass and heat flow), and the processed material, but the die design (particularly the die diameter) is also highly important. To ensure high porosity of the extrudates, a high temperature is required, usually from 140 to 160 °C . OperatiPolyphenols are secondary metabolites of plants that are used in their defense against severe environments, such as ultraviolet radiation or attack by pathogens . These cPhenolic compounds have two forms—free and bound. Bound phenolic compounds are often bound with cell wall components . DecompoHirth et al.  showed tThe total phenolic content of extrudates is stated in numerous scientific papers. Thus, in the current study we focused more on individual groups of phenolics  and their changes during extrusion cooking. The object of the study was to compare analytical results in premixes and extrudates (retention of phenolics). We are aware that mixing results of TPC/TFC with other specific determinations (chromatography/spectrometry coupled methods) is not always correct, but the data provide basic information for new food design. The paper does not address the suitability of the analytical methods used.Application of one-screw or twin-screw extrusion is also investigated in this review. The detailed arrangement of the instruments and sensory properties of the products can be found in the reference papers.The first step in the design of a new product should be trials of the extrusion processing for a one-component blend, although the input blend clearly does not have a single component ; the second component is water. Sensory properties of the resulting extrudates  depend foremost on the quantity of water used. The single component data enables the impacts on TPC to be assessed in real mixtures.The TPC data of one-component extrudates from recent years are included in Corn extrudates have special properties and are of economic importance in the food industry. It is clear from Many data have been published for rice flour, particularly for brown rice. Different results of TPC for extrudates prepared by a single-screw extruder compared to a twin-screw extruder were found in the ranges of 94–178 and 46–77 mg/100 g DM, respectively. It is likely that stronger conditions in a single-screw extruder caused higher disruption of cell wall matrices. Besides extrusion conditions, processing temperature and screw speed are also important. Other data were measured for flour from polished rice, broken rice, glutinous rice, glutinous rice with α-amylase addition, extruded soaked rice, and extruded germinated rice. As expected, the highest value was found for rice bran—811 mg/100 g DM.The data for wheat in Sorghum is a gluten-free cereal that has the highest content of phenolic compounds among cereals but must be processed prior to use for human consumption. The phenolic compounds of sorghum, including proanthocyanidins, 3-deoxyanthocyanidins, and flavones, beneficially modulate the gut microbiota and variables related to noncommunicable diseases, such as obesity, diabetes, dyslipidemia, cardiovascular disease, and cancer . TPC in Chenopodium, dried mango, tigernut flour, dried green banana flour, and millet. The highest TPC values have been found in extrudates of oven-dried green banana flour, tigernut flour, and soybean flour, having values of 1196, 1000, and 652 mg/100 g DM, respectively.Other data in Numerous TPC data of extrudates are presented in the literature for which, in addition to water and one main component, additives were also used, e.g., materials rich in phenolics and/or waste from the food industry. This procedure enables important phenolics in extrudates to be increased. Therefore, we also complied interesting TPC data for blends comprising a greater number of components and their extrudates in Phenolic acids are a highly important component of phenolics, e.g., phenolics present in cereals are mostly phenolic acids covalently bound to the cell wall, together with carotenoids, tocopherols, and tocotrienols . Newly pNew laboratory techniques such as (ultra) high-performance liquid chromatography (HPLC) electrospray ionization tandem (quadrupole time-of-flight) mass spectrometry, and the simple HPLC system, have enabled phenolic acids to be separately identified.Dracocephalum moldavica L.) leaves in extruded corn snacks. The data of the individual phenolic acids from the literature in The individual data from the literature are also numerous. Therefore The largest increase (retention) was found for sinapic acid in various materials: 262% in oat flour, 275% in corn grit, and 405% in brown rice flour. However, the final concentration in extrudates was not high: between 2 and 9 mg/100 g DM. Although some authors have indicated that wine is a good source of sinapic acid, the concentration is only about 0.2–0.4 mg/L , and higZhang et al.  detectedFurthermore, Zeng et al.  and GongCocos nucifera, having 44 mg/100 g DM [4-OH-benzoic acid shows antimicrobial activity, and has significant retention (214%) in oat flour extrudates where the final content was about 1 mg/100 g DM. This value is very low compared with the uncharacteristic predominance of 4-OH-benzoic acid in the mesocarp of 100 g DM .Lohani and Muthukumarappan  extrudedBrassica oleracea L. . Both the qualitative and quantitative contents of phenolics increased with the addition of kale; 3-OH-cinnamic acid occurred only in snacks enriched with 6% kale. Oniszczuk et al. [Kasprzak et al.  preparedk et al.  processek et al.  investigSignificant degrees of antioxidant and anti-tumor activity and the converse, carcinogenicity, have been attributed to flavonoids, and it is their effect on human health which has brought them to wider attention. The basic structure of the major groups of flavonoids can be found in the literature . Table 4The most well-known flavonoids are anthocyans, flavanons, flavonols, and chalcones . FlavonoThe content of total flavonols after extrusion increased in apple pomace  but, conThe phenolic profile of flavonols was determined using the Dionex Ultimate 3000 UPLC , where data were collected simultaneously with a diode array detector (280 and 370 nm) and a mass spectrometer  operating in negative mode ,76. The According to Liu et al. , quercetProanthocyanidins are oligomers or polymers of flavan-3-ols and are often found in fruits, berries, beans, nuts, cocoa, and wine. Proanthocyanidins are considered to have a wide range of biological properties, including antioxidant, anticarcinogenic, cardioprotective, antimicrobial, and neuroprotective activities, as demonstrated by a number of in vitro and in vivo studies . CardosoAzad et al.  preparedExtrusion cooking induces the alteration of raw materials in physicochemical and functional properties, phenolic compounds, and their antioxidant activities. Decomposition of heat-labile phenolic compounds and polymerization during extrusion tend to decrease the extractable phenolic content. In contrast, due to the disruption of cell wall matrices and the breaking of high molecular weight complex phenolics during extrusion, the extractability of phenolic compounds is improved.Numerous interesting data relating to phenolics have been published for rice flour, particularly that of brown rice, and composite mixtures. The largest values of phenolic acids in extrudates were found in rice bran. Composite mixtures include, e.g., addition of orange peel powder, green tea, goji berries, bean grits, açaí, and carrot. High levels of TPC, of 3285–4038 and 1522–3160 mg/100 g DM, were found in extrudates of soy protein isolate/wheat gluten/corn starch/green tea and lentil flour/orange peels powder, respectively.Dracocephalum moldavica, Cocos nucifera, Brassica oleracea, red potatoes (Solanum tuberosum var. Magenta Love), strawberries (Fragariaananassa), and apple pomace, can achieve even higher concentrations of selected phenolic acids.Regarding phenolic acids, excellent sources of vanillic acid and syringic acid are extrudates based on rice bran or wheat flour. The use of additives in the cereal premix, e.g., Extrusion reduces flavonoids by 40% and 30% in polished rice and brown rice, respectively. The flavonol content after extrusion was found to increase in apple pomace, but decrease in lentil flour with retention of only 13%. Regarding flavonol content in extrudates, we recommend that new experiments should be conducted with rice or lentil flour mixed with parsley or dill. Flavanones, flavones, and 3-deoxyanthocyanidins in the extruded genotypes of sorghum are destroyed after extrusion cooking due to their thermal sensitivity.Thus, extrusion cooking can improve or worsen the digestibility of phenolic compounds. Higher values of specific phenolic compounds in extrudates create the potential for new food products.


Supplemental Digital Content is available in the text. Pseudonaja textilis venom-derived factor V) variant retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of FVa .The Australian snake venom ptFV (Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic.Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity. Pseudonaja textilis retains cofactor function despite proteolysis by factor Va’s natural inhibitor.Venom-derived factor Va from the Australian snake Whereas the inactivation of human factor Va proceeds via A2 domain dissociation following limited proteolysis, A2 domain dissociation is prevented by strong noncovalent interactions in venom-derived factor Va, allowing the persistent conversion of prothrombin into thrombin.Molecular dynamics simulations revealed that venom-derived factor Va comprises a rigid loop in the A2 domain that forms productive interactions with the A1 domain. These interactions increase the binding affinity between the A1 and A2 domains, whereas the weak interactions in human factor Va result in A2 domain dissociation upon proteolysis.3 Once FV is partially or fully activated, it functions as cofactor for the serine protease FXa (factor Xa) by formation of the prothrombinase complex on the surface of an anionic membrane in the presence of calcium ions.5 This complex rapidly converts prothrombin to thrombin, a key regulatory enzyme in coagulation. The coagulation system can also be exploited to gain selective advantages. The procoagulant venom of several Australian Elapid snakes contains a powerful enzyme complex that specifically activates prothrombin to disrupt the prey’s intrinsic hemostatic balance.9 This prothrombin-activating complex consists of FX- and FV-like proteins13 comprising certain remarkable gain-of-function adaptations that enable this prothrombinase-like complex to initiate coagulation in an uncontrolled manner.17 Rao et al11 and our group18 have demonstrated that common brown snake venom ptFV (Pseudonaja textilis venom-derived factor V), while undergoing selective proteolysis, is functionally resistant to FVa’s natural inhibitor APC (activated protein C).Blood coagulation is a tightly regulated process composed of the sequential and concerted action of several serine proteases and their respective cofactors, together responsible for minimizing blood loss following vascular injury. Central to this process is the activation of FV (coagulation factor V) into the active FVa (cofactor Va).See cover image23 Cleavage at Arg306 and Arg506 results in A2 domain dissociation and subsequent loss of FXa affinity.24 While ptFVa is cleaved at a position that is homologous to human Arg506 (Lys507),14 an Arg306-like cleavage site is absent.14 Furthermore, venom FV comprises a unique disulfide bond that covalently links the A2 and A3 domains.16 Although we have previously reported that this disulfide bond is not responsible for the functional APC resistance,18 whether it might stabilize the A2 domain binding affinity upon introduction of the Arg306 cleavage site remains to be determined.To downregulate the procoagulant response, coagulant FVa is inactivated by APC via proteolysis of the A2 domain residues Arg306, Arg506, and Arg679.In the present study, we generated several chimeric ptFV variants with the goal of uncovering the mechanistic principles of its exceptional functional resistance to APC. Our results demonstrate that ptFV has uniquely adapted several structural elements to prevent A2 domain dissociation and ptFVa inactivation. As such, these findings highlight the remarkable evolutionary adaptations that have made ptFVa into an extremely stable procoagulant venom and provide new insights in the structural requirements of APC-induced FVa inactivation.The data that support the findings of this study are available from the corresponding author upon reasonable request.25 FV-depleted human plasma and Neoplastine CI Plus 10 prothrombin time reagent were obtained from Diagnostica Stago . All functional assays were performed in HEPES buffered saline  supplemented with 5 mmol/L CaCl2, 0.1% PEG8000, and filtered over a 0.2 µm filter (assay buffer).The inhibitor dansylarginine-N-amide was from Haematologic Technologies . The peptidyl substrate H-D-Phe-Pip-Arg-pNA (S2238) was obtained from Instrumentation Laboratories . All tissue culture reagents were from Life Technologies . Small unilamellar phospholipid vesicles (PCPS) composed of 75% (wt/wt) hen egg L-phosphatidylcholine and 25% (wt/wt) porcine brain L-phosphatidylserine  were prepared and characterized as described previously.Pseudonaja textilis venom-derived factor Xa) was prepared, purified, and characterized as described.18 Recombinant constitutively active B-domainless hFV  and venom-derived ptFV were prepared, purified, and characterized as described previously.26 The molecular weight and the extinction coefficients  of the newly generated hFV-pt306, ptFV-h306, and ptFV-h306 S-S were assumed to be equal to hFV  and ptFV , respectively.26The human plasma-derived coagulation factors FXa, prothrombin, α-thrombin, APC, protein S, and plasmin were from Haematologic Technologies . Restriction endonucleases XcmI and Bsu36I were obtained from New England Biolabs . Recombinant venom-derived ptFXa  was removed by replacing the cysteines for serine residues, thereby creating pED-ptFV-h306 S-S. ptFV-h306 and ptFV-h306 S-S were generated employing mutagenic complementary oligonucleotides as described.14 The variant hFV-pt306 was generated using a pUC57 plasmid encoding pED-ptFV nucleotides 2124-2144, flanked by pED-hFV nucleotides, which was generated and purchased from Genscript . The resulting pUC57 plasmid was digested with XcmI and Bsu36I, gel-purified, and subcloned into pED-hFV via digestion with the same enzymes.Constructs encoding for hFV comprising the ptFV Arg306 region , designated pED-hFV-pt306, and ptFV comprising the human Arg306 region sequence , named pED-ptFV-h306 were generated. Subsequently, ptFV’s unique disulfide bond  or SP-Sepharose (ptFV variants) column  using a NaCl elution gradient in HEPES buffered saline supplemented with 5 mmol/L CaCl2 and 1 mmol/L benzamidine, pH 7.4. Fractions containing FV activity were pooled and dialyzed against 20 mmol/L HEPES, 5 mmol/L CaCl2, pH 7.4 for human FV variants or 20 mmol/L MES, 5 mmol/L CaCl2, pH 6.0 for ptFV variants, loaded on a POROS HQ/20 (hFV variants), or HS/20 column  and eluted using a NaCl gradient. Protein purity was assessed by SDS-PAGE analysis using precast 4% to 12% gradient gels under nonreducing and reducing conditions (50 mmol/L dithiothreitol) using the MOPS buffer system  followed by staining with Coomassie Brilliant Blue R-250. For pretreatment with thrombin, FV variants were incubated for 15 minutes with α-thrombin  in assay buffer at 37 °C, followed by the addition of a 10-fold molar excess of hirudin over thrombin.Transfection of the plasmids encoding FV variants into baby hamster kidney cells, the selection of stable clones, and the expression and purification of hFV and ptFV were performed as described previously.28 Briefly, unless otherwise stated, progress curves of prothrombin were obtained by incubating PCPS (50 μmol/L), dansylarginine-N-amide (10 µmol/L), and prothrombin (1.4 µmol/L) with the various recombinant FV(a) species (0-40 nmol/L), and the reaction was initiated with 0.4 nmol/L of FXa or ptFXa upon which the rate of prothrombin conversion was measured.27Steady-state initial velocities of macromolecular substrate cleavage were determined discontinuously at 25 °C in assay buffer as described.27 Identification of the APC cleavage sites by N-terminal sequence analysis was performed at The Protein Facility of the Iowa State University Office of Biotechnology .For APC-mediated proteolysis, FVa variants (500 nmol/L) were first incubated at 37 °C with APC  in the presence of PCPS (50 µmol/L). In addition, similar reactions containing ptFVa variants (50 nmol/L) and PCPS (50 µmol/L) were treated with 75 nmol/L APC in the presence of protein S (100 nmol/L). Following APC treatment, aliquots were taken and assayed for cofactor activity. Activity assay mixtures contained prothrombin (1.4 µmol/L), PCPS (50 µmol/L), dansylarginine-N-amide (10 µmol/L), APC-proteolyzed hFVa (0.2 nmol/L) or ptFVa variants (0.1 nmol/L), and FXa (2 nmol/L) or ptFXa (1 nmol/L), respectively. Prothrombin activation was determined as described.−1 at room temperature. Fractions (0.25 mL) were collected and analyzed by SDS-PAGE or Western Blot using a polyclonal antibody directed against human FV  and the monoclonal antibody directed against the Asn307-Arg506 fragment  as described.29FVa variants (100–500 µg) were subjected to APC treatment  for 15 (hFVa) or 180 minutes (ptFVa) at 37 °C in the presence of 50 µmol/L PCPS. Reactions were quenched by the addition of 50 µmol/L PPACK and 1 mmol/L benzamidine. A 10×300 mm column containing Superdex 200 Increase  was equilibrated in assay buffer supplemented with 50 µmol/L PPACK and 1 mmol/L benzamidine. The 100 to 400 µL reaction mixture was then applied to the column and eluted with equilibration buffer at a flow rate of 0.25 mL min30 the 3-dimensional structures of hFVa31 and ptFV (PDB ID: 4BXS) were used to build in missing loops with Loop Modeling suite in YASARA.32 In addition, to simulate the experimentally used ptFV variant, the original region (303-GNPDTLT-309) was exchanged for the human Arg306 region (302-PKKTRNL-308), and the unique Cys642-Cys1002 disulfide bond was disrupted. To mimic the APC-cleaved protein structure, both human and P. textilis FV were separated into 2 protein fragments, reflecting their state after cleavage by APC at Arg306 and Arg506/Lys507. To expedite simulations, the C1 and C2 domain structures were excluded from simulation for both the human and P. textilis FV structures, given that these domains are structurally nonrelated with the A2 domain.For molecular dynamics (MD) simulation,− or Na+ ions. The topology and coordinate files were prepared with the Amber ff14SB33 force field. During MD simulations, 2 initial energy minimization runs were performed using a steepest descent algorithm of 50 000 steps. Subsequently, each system was gradually heated from 0 to 300 K within 50 picoseconds. This step was followed by another 50 picoseconds equilibration run with a constant temperature of 300 K. After that, a 100 ns production run was carried out using the SHAKE algorithm34 to constrain bonds involving hydrogen atoms. The minimization, heating, equilibration, and production runs were all applied using AMBER 16.35Each simulation system was solvated with an explicit TIP3P water model and neutralized by addition of Clt=0 to 20 ns and t=80 to 100 ns, to calculate binding free energies. The Molecular Mechanics/Generalized Born Surface Area method36 was successfully employed using igb=5 GB model in AMBER, which can be briefly summarized by the following equations:From the 100 ns production run trajectory, 2000 frames were extracted between int, Eele, and EvdW representing internal, electrostatic and van der Waals interactions; the solvation contribution is shown with Epolar and Enonpolar. The TΔS term represents the contribution of entropy which was not taken into account when calculating relative binding free energies in our study. To determine key residues involved in binding, a per-residue energy contribution was calculated and analyzed based on the same factors used in above calculations.The binding free energy was calculated as the differences between free energy of the complex and the sum of the separate receptor and ligand shown in Equation 1. The relative free energy ΔGMM/GBSA was estimated by the sum of every component in Equation 2, with EP value of <0.05. All data are presented as mean±1 SD and are the result of at least 2 to 3 independent experiments, unless otherwise stated.All statistical analyses were computed using the GraphPad Prizm software. Kinetic data were analyzed by nonlinear regression using a 3-parameter logistic function or an 1-phase decay function. Statistical significance was accepted for 14 APC cleaves the ptFV A2 domain at Lys507 and Arg742, homologous to human Arg506 and Arg709, respectively. Intriguingly, sequence analysis revealed the lack of a potential Arg306 cleavage site and a nonconserved sequence relative to mammalian FV consisting of ptFV residues 303-GNPDTLT-309 , indicating that substitution of the Arg306 region does not interfere with cofactor-FXa assembly.Previously, we reported that ptFV retains full cofactor activity despite APC-catalyzed proteolysis.24 Thrombin-activated FVa variants were subjected to APC, and the resulting cleavage pattern was analyzed by SDS-PAGE. While in hFVa variants full proteolysis was achieved in 15 minutes by 10 nmol/L APC, high concentrations of APC (750 nmol/L) were required for significant proteolysis of the ptFVa variants. As previously described,14 ptFVa was cleaved at Lys507 , which is equivalent to human Arg506. Conversely, we observed additional protein bands consistent with the cleavage of Arg306 in ptFVa-h306 . Removal of the unique disulfide bond in ptFVa-h306 S-S did not affect the cleavage pattern relative to ptFVa-h306 . Moreover, while hFVa was proteolyzed at Arg306, Arg506, and Arg679 , an Arg306-like cleavage was absent in hFVa-pt306 . Intriguingly, our results show that nonproteolyzed hFV migrated as multiple species. Although the individual species could not be identified, this observation might be explained by the presence of impurities  or high ionic strength  did not reveal significant differences relative to wild-type FVa, suggestive of an enhanced A2 domain binding stability. Interestingly, proteolysis by the fibrinolytic serine protease plasmin revealed a striking decrease in ptFVa cofactor activity . While the loss of cofactor activity was comparable to wild-type FVa, all ptFVa variants retained up to 20% cofactor activity despite that full proteolysis was achieved within 5 minutes . These findings provide additional support for an enhanced structural stability of essential functional regions in ptFVa.APC-catalyzed proteolysis at the 2 major FVa cleavage sites Arg306 and Arg506 is known to result in A2 domain dissociation.P. textilis FV, 100 ns MD simulations were performed on hFVa and ptFVa-h306 S-S. The model of ptFVa-h306 S-S was generated from the X-ray structure of ptFV (PDB ID: 4BXS) in which 303-GNPDTLT-309 was substituted for the corresponding human Arg306 region and the Cys642-Cys1002 disulfide bond was disrupted. In addition, the peptide bonds that resemble Arg306 and Arg506/Lys507 cleavage were hydrolyzed in both FVa models to simulate an APC-proteolyzed state, and the C1-C2 domains were excluded from the MD simulations. Calculation of the free energies for the interaction between the A2 and A1-A3 domains revealed that these were comparable for hFVa and ptFVa-h306 S-S during the initial trajectory of the simulation (t=0–20 ns), with a binding free energy of −76±25 kcal/mol for hFVa and −76±14 kcal/mol for ptFVa-h306 S-S (Table t=80–100 ns), the binding free energy was remarkably increased to −39±16 kcal/mol for hFVa, whereas a substantial decrease to −90±14 kcal/mol was observed for ptFVa-h306 S-S. This suggests that over the course of the MD simulation, hFVa lost important interactions that facilitate the binding of the A2 to the A1-A3 domains. To examine these interdomain interactions in more detail, the binding free energies between the A2 and A1 or A2 and A3 domains were individually assessed. Interestingly, a loss of interaction between the A2 and A1 domains in hFVa was observed for the t=80−100 ns interval . Collectively, these findings indicate that ptFV comprises a high number of A2-A1 interactions that are preserved upon APC cleavage.To further uncover the molecular mechanisms that are at the basis of the differential A2-A1 domain interactions observed for hFV and ptFV, the hydrogen bonds that contribute to the overall interdomain binding affinities were assessed. In accordance with the loss of A2-A1 domain interaction, the number of hydrogen bonds between the surfaces of the A2 and A1 domain in hFVa reduced over time during the MD simulations Figure A. RepresData Supplement). While human and P. textilis FVa share 55% sequence identity, the positioning of loop1 and loop2 is conserved . In addition, sequence analysis showed that loop2 is well-conserved in reptiles, whereas a significantly different sequence is conserved in mammalian FV . Interestingly, MD simulations in ptFVa-h306 S-S indicated that loop2 is more compact and does not interact with loop1 . This was found to be the result of a more compact A2 domain, as compared with the A1-A3 domains. To specifically determine the role of loop2 in A2 domain stability, we created an in silico chimeric hFVa structure in which the human loop2 was exchanged for the homologous ptFV loop2 region (hFVa-ptloop2), upon which a 100 ns MD simulation was performed  as compared with hFVa (16689.3±0.04 Å2), but remained larger relative to ptFVa-h306 S-S (14761.8±0.04 Å2), illustrating the compact conformation of the ptFV loop2. Consistent with these findings, the A2-A1 domain hydrogen bonds were stabilized in chimeric hFVa-ptloop2, while the exchange of loop2 did not affect the interactions between the A2 and A3 domains , Landsteiner Foundation for Blood Transfusion , and the China Scholarship Council (to X. Liu). The funding agencies had no role in the preparation, review, or approval of the article.P.H. Reitsma owns equity in VarmX B.V. The other authors report no conflicts.