Differences in the Membrane-Binding Properties of Flaviviral Nonstructural 1 (NS1) Protein: Comparative Simulations of Zika and Dengue Virus NS1 Proteins in Explicit Bilayers.

NS1 in flaviviruses is the only nonstructural protein that is secretory and interacts with different cellular components of the host cell membrane. NS1 is localized in the ER as a dimer to facilitate viral replication. Crystal structures of NS1 homologues from zika (ZIKV) and dengue (DENV) viruses have revealed the organization of different domains in NS1 dimers. The ß-roll and the connector and intertwined loop regions of wing domains of NS1 have been shown to interact with the membranes. In this study, we have performed multiple molecular dynamics (MD) simulations of ZIKV and DENV NS1 systems in apo and in POPE bilayers with different cholesterol concentrations (0, 20 and 40%). The NS1 protein was placed just above the membrane surface, and for each NS1-membrane system two to three independent simulations with 600 ns production run were performed. At the end of the production runs, ZIKV NS1 inserts deeper inside the membrane compared to the DENV counterpart. Unlike ZIKV NS1, the orientation of DENV NS1 is asymmetric in which one of the chains in the dimer interacts with the membrane while the other is more exposed to the solvent. The ß-roll region in ZIKV NS1 penetrates beyond the headgroup region and interacts with the lipid acyl chains while the C-terminal region barely interacts with the headgroup. Specific residues in the intertwined region deeply penetrate inside the membrane. The role of charged and aromatic residues of ZIKV NS1 in strongly interacting with the membrane components is revealed. The presence of cholesterol affects the extent of insertion in the membrane and interaction of individual residues. Overall, membrane-binding properties of ZIKV NS1 significantly differ from its counterpart in DENV. The differences found in the binding and insertion of NS1 can be used to design drugs and novel antibodies that can be flavivirus specific.

Crystal Engineering and Self-Assembled Nanoring Formation with Purine-CdII/HgII Supramolecular Frameworks.

We report three complexes of CdII and HgII with two purine rare tautomers, N9-(pyridin-2-ylmethyl)-N6-methoxyadenine, L1 and N7-(pyridin-2-ylmethyl)-N6-methoxyadenine, L2, highlighting diverse crystallographic signatures exhibited by them. Influence of substituents, binding sites, steric effects and metal salts on the different modes of binding enabled an insight into metal-nucleobase interactions. L1 interacted with two and three equivalents of Cd(NO3)2.4H2O and HgCl2, respectively, while L2 interacted with two equivalents of HgCl2, altogether leading to three different complexes (1 [C48H48Cd6N34O50], 2 [C12H12Cl4Hg2N6O] and 3 [C12H12Cl2HgN6O]) possessing varied dimensionality and stabilising interactions. The photoluminescent properties of these coordination frameworks have also been probed. Notably, nanoring-like structures were obtained, as a result of self-assembly of 3 when investigated by transmission electron microscopy, additionally supported by molecular dynamics simulations.

Substrate selectivity and unique sequence signatures in SWEET/semiSWEET homologs of four taxonomic groups: Sequence analysis and phylogenetic studies.

The recently discovered SWEET (Sugar Will Eventually be Exported Transporter) proteins are involved in the selective transport of monosaccharides and disaccharides. The prokaryotic counterparts, semiSWEETs, form dimers with each monomer forming a triple-helix transmembrane bundle (THB). The longer eukaryotic SWEETs have seven transmembrane helices with two THBs and a linker helix. Structures of semiSWEETs/SWEETs have been determined experimentally. Experimental studies revealed the role of plant SWEETs in vital physiological processes and identified residues responsible for substrate selectivity. However, SWEETs/semiSWEETs from metazoans and bacteria are not characterized. In this study, we used structure-based sequence alignment and compared more than 2000 SWEET/semiSWEETs from four different taxonomic groups. Conservation of residue/chemical property was examined at all positions. Properties of clades/subclades of phylogenetic trees from each taxonomic group were analyzed. Conservation pattern of known residues in the selectivity-filter was used to predict the substrate preference of plant SWEETs and some clusters of metazoans and bacteria. Some residues at the gating and substrate-binding regions, pore-facing positions and at the helix-helix interface are conserved across all taxonomic groups. Conservation of polar/charged residues at specific pore-facing positions, helix-helix interface and in loops seems to be unique for plant SWEETs. Overall, the number of conserved residues is less in metazoan SWEETs. Plant and metazoan SWEETs exhibit high conservation of four and three proline residues respectively in "proline tetrad." Further experimental studies can validate the predicted substrate selectivity and significance of conserved polar/charged/aromatic residues at structurally and functionally important positions of SWEETs/semiSWEETs in plants, metazoans and bacteria.

dbAQP-SNP: a database of missense single-nucleotide polymorphisms in human aquaporins.

Aquaporins and aquaglyceroporins belong to the superfamily of major intrinsic proteins (MIPs), and they transport water and other neutral solutes such as glycerol. These channel proteins are involved in vital physiological processes and are implicated in several human diseases. Experimentally determined structures of MIPs from diverse organisms reveal a unique hour-glass fold with six transmembrane helices and two half-helices. MIP channels have two constrictions formed by Asn-Pro-Ala (NPA) motifs and aromatic/arginine selectivity filters (Ar/R SFs). Several reports have found associations among single-nucleotide polymorphisms (SNPs) in human aquaporins (AQPs) with diseases in specific populations. In this study, we have compiled 2798 SNPs that give rise to missense mutations in 13 human AQPs. To understand the nature of missense substitutions, we have systematically analyzed the pattern of substitutions. We found several examples in which substitutions could be considered as non-conservative that include small to big or hydrophobic to charged residues. We also analyzed these substitutions in the context of structure. We have identified SNPs that occur in NPA motifs or Ar/R SFs, and they will most certainly disrupt the structure and/or transport properties of human AQPs. We found 22 examples in which missense SNP substitutions that are mostly non-conservative in nature have given rise to pathogenic conditions as found in the Online Mendelian Inheritance in Man database. It is most likely that not all missense SNPs in human AQPs will result in diseases. However, understanding the effect of missense SNPs on the structure and function of human AQPs is important. In this direction, we have developed a database dbAQP-SNP that contains information about all 2798 SNPs. This database has several features and search options that can help the user to find SNPs in specific positions of human AQPs including the functionally and/or structurally important regions. dbAQP-SNP (http://bioinfo.iitk.ac.in/dbAQP-SNP) is freely available to the academic community. Database URL http://bioinfo.iitk.ac.in/dbAQP-SNP.

Molecular dynamics studies of CED-4/CED-9/EGL-1 ternary complex reveal CED-4 release mechanism in the linear apoptotic pathway of Caenorhabditis elegans.

Many steps in programmed cell death are evolutionarily conserved across different species. The Caenorhabditis elegans proteins CED-9, CED-4 and EGL-1 involved in apoptosis are respectively homologous to anti-apoptotic Bcl-2 proteins, Apaf-1 and the "BH3-only" pro-apototic proteins in mammals. In the linear apoptotic pathway of C. elegans, EGL-1 binding to CED-9 leads to the release of CED-4 from CED-9/CED-4 complex. The molecular events leading to this process are not clearly elucidated. While the structures of CED-9 apo, CED-9/EGL-1 and CED-9/CED-4 complexes are known, the CED-9/CED-4/EGL-1 ternary complex structure is not yet determined. In this work, we modeled this ternary complex and performed molecular dynamics simulations of six different systems involving CED-9. CED-9 displays differential dynamics depending upon whether it is bound to CED-4 and/or EGL-1. CED-4 exists as an asymmetric dimer (CED4a and CED4b) in CED-9/CED-4 complex. CED-4a exhibits higher conformational flexibility when simulated without CED-4b. Principal Component Analysis revealed that the direction of CED-4a's winged-helix domain motion differs in the ternary complex. Upon EGL-1 binding, majority of non-covalent interactions involving CARD domain in the CED-4a-CED-9 interface have weakened and only half of the contacts found in the crystal structure between α/ß domain of CED4a and CED-9 are found to be stable. Additional stable contacts in the ternary complex and differential dynamics indicate that winged-helix domain may play a key role in CED-4a's dissociation from CED-9. This study has provided a molecular level understanding of potential intermediate states that are likely to occur when CED-4a is released from CED-9.

Designing BH3-Mimetic Peptide Inhibitors for the Viral Bcl-2 Homologues A179L and BHRF1: Importance of Long-Range Electrostatic Interactions.

Viruses have evolved strategies to prevent apoptosis of infected cells at early stages of infection. The viral proteins (vBcl-2s) from specific viral genes adopt a helical fold that is structurally similar to that of mammalian antiapoptotic Bcl-2 proteins and exhibit little sequence similarity. Hence, vBcl-2 homologues are attractive targets to prevent viral infection. However, very few studies have focused on developing inhibitors for vBcl-2 homologues. In this study, we have considered two vBcl-2 homologues, A179L from African swine fever virus and BHRF1 from Epstein-Barr virus. We generated two sets of 8000 randomized BH3-like sequences from eight wild-type proapoptotic BH3 peptides. During this process, the four conserved hydrophobic residues and an Asp residue were retained at their respective positions, and all other positions were substituted randomly without any bias. We constructed 8000 structures each for A179L and BHRF1 in complex with BH3-like sequences. Histograms of interaction energies calculated between the peptide and the protein resulted in negatively skewed distributions. The BH3-like peptides with high helical propensities selected from the negative tail of the respective interaction energy distributions exhibited more favorable interactions with A179L and BHRF1, and they are rich in basic residues. Molecular dynamics studies and electrostatic potential maps further revealed that both acidic and basic residues favorably interact with A179L, while only basic residues have the most favorable interactions with BHRF1. As in mammalian homologues, the role of long-range interactions and nonhotspot residues has to be taken into account while designing specific BH3-mimetic inhibitors for vBcl-2 homologues.

Computational Design of BH3-Mimetic Peptide Inhibitors That Can Bind Specifically to Mcl-1 or Bcl-XL: Role of Non-Hot Spot Residues.

Interactions between pro- and anti-apoptotic Bcl-2 proteins decide the fate of the cell. The BH3 domain of pro-apoptotic Bcl-2 proteins interacts with the exposed hydrophobic groove of their anti-apoptotic counterparts. Through their design and development, BH3 mimetics that target the hydrophobic groove of specific anti-apoptotic Bcl-2 proteins have the potential to become anticancer drugs. We have developed a novel computational method for designing sequences with BH3 domain features that can bind specifically to anti-apoptotic Mcl-1 or Bcl-XL. In this method, we retained the four highly conserved hydrophobic and aspartic residues of wild-type BH3 sequences and randomly substituted all other positions to generate a large number of BH3-like sequences. We modeled 20000 complex structures with Mcl-1 or Bcl-XL using the BH3-like sequences derived from five wild-type pro-apoptotic BH3 peptides. Peptide-protein interaction energies calculated from these models for each set of BH3-like sequences resulted in negatively skewed extreme value distributions. The selected BH3-like sequences from the extreme negative tail regions have highly favorable interaction energies with Mcl-1 or Bcl-XL. They are enriched in acidic and basic residues when they bind to Mcl-1 and Bcl-XL, respectively. With the charged residues often away from the binding interface, the overall electric field generated by the charged residues results in strong long-range electrostatic interaction energies between the peptide and the protein giving rise to high specificity. Cell viability studies of representative BH3-like peptides further validated the predicted specificity. This study has revealed the importance of non-hot spot residues in BH3-mimetic peptides in providing specificity to a particular anti-apoptotic protein.

Is the E. coli Homolog of the Formate/Nitrite Transporter Family an Anion Channel? A Computational Study.

Formate/nitrite transporters (FNTs) selectively transport monovalent anions and are found in prokaryotes and lower eukaryotes. They play a significant role in bacterial growth and act against the defense mechanism of infected hosts. Because FNTs do not occur in higher animals, they are attractive drug targets for many bacterial diseases. Phylogenetic analysis revealed that they can be classified into eight subgroups, two of which belong to the uncharacterized YfdC-α and YfdC-ß groups. Experimentally determined structures of FNTs belonging to different phylogenetic groups adopt the unique aquaporin-like hourglass helical fold. We considered the formate channel from Vibrio cholerae, the hydrosulphide channel from Clostridium difficile, and the uncharacterized channel from Escherichia coli (EcYfdC) to investigate the mechanism of transport and selectivity. Using equilibrium molecular dynamics and umbrella sampling studies, we determined temporal channel radius profiles, permeation events, and potential of mean force profiles of different substrates with the conserved central histidine residue in protonated or neutral form. Unlike the formate channel from V. cholerae and the hydrosulphide channel from C. difficile, molecular dynamics studies showed that the formate substrate was unable to enter the vestibule region of EcYfdC. Absence of a conserved basic residue and presence of acidic residues in the vestibule regions, conserved only in YfdC-α, were found to be responsible for high energy barriers for the anions to enter EcYfdC. Potential of mean force profiles generated for ammonia and ammonium ion revealed that EcYfdC can transport neutral solutes and could possibly be involved in the transport of cations analogous to the mechanism proposed for ammonium transporters. Although YfdC members belong to the FNT family, our studies strongly suggest that EcYfdC is not an anion channel. Absence or presence of specific charged residues at particular positions makes EcYfdC selective for neutral or possibly cationic substrates. Further experimental studies are needed to get a definitive answer to the question of the substrate selectivity of EcYfdC. This provides an example of membrane proteins from the same family transporting substrates of different chemical nature.

Presence of Intra-helical Salt-Bridge in Loop E Half-Helix Can Influence the Transport Properties of AQP1 and GlpF Channels: Molecular Dynamics Simulations of In Silico Mutants.

Major intrinsic protein (MIP) superfamily contains water-transporting AQP1 and glycerol-specific GlpF belonging to two major phylogenetic groups, namely aquaporins (AQPs) and aquaglyceroporins (AQGPs). MIP channels have six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). LE region contributes two residues to the aromatic/arginine (Ar/R) selectivity filter (SF) within the MIP channel. Bioinformatics analyses have shown that all AQGPs have an intra-helical salt-bridge (IHSB) in LE half-helix and all AQGPs and majority of AQPs have helix destabilizing Gly and/or Pro in the same region. In this paper, we mutated in silico the acidic and basic residues in GlpF to Ser and introduced salt-bridge interaction in AQP1 LE half-helix by substituting Ser residues at the equivalent positions with acidic and basic residues. We investigated the influence of IHSB in LE half-helix on the transport properties of GlpF and AQP1 mutant channels using molecular dynamics simulations. With IHSB abolished in LE half-helix, the GlpF mutant exhibited a significantly reduced water transport. In contrast, the introduction of IHSB in the two AQP1 mutants has increased water transport. Absence of salt-bridge in LE half-helix alters the SF geometry and results in a higher energy barrier for the solutes in the Ar/R selectivity filter. Presence/absence of IHSB in LE half-helix influences the channel transport properties and it is evident especially for the AQGPs. By modulating its helical flexibility, LE half-helix can perhaps play a regulatory role in transport either on its own or in conjunction with other extracellular regions.

Cooperativity in Plant Plasma Membrane Intrinsic Proteins (PIPs): Mechanism of Increased Water Transport in Maize PIP1 Channels in Hetero-tetramers.

Plant aquaporins (AQPs) play vital roles in several physiological processes. Plasma membrane intrinsic proteins (PIPs) belong to the subfamily of plant AQPs. They are further subdivided into two closely related subgroups PIP1s and PIP2s. While PIP2 members are efficient water channels, PIP1s from some plant species have been shown to be functionally inactive. Aquaporins form tetramers under physiological conditions. PIP2s can enhance the water transport of PIP1s when they form hetero-tetramers. However, the role of monomer-monomer interface and the significance of specific residues in enhancing the water permeation of PIP1s have not been investigated at atomic level. We have performed all-atom molecular dynamics (MD) simulations of homo-tetramers and four different hetero-tetramers containing ZmPIP1;2 and ZmPIP2;5 from Zea mays. ZmPIP1;2 in a tetramer assembly will have two interfaces, one formed by transmembrane segments TM4 and TM5 and the other formed by TM1 and TM2. We have analyzed channel radius profiles, water transport and potential of mean force profiles of ZmPIP1;2 monomers. Results of MD simulations clearly revealed the influence of TM4-TM5 interface in modulating the water transport of ZmPIP1;2. MD simulations indicate the importance of I93 residue from the TM2 segment of ZmPIP2;5 for the increased water transport in ZmPIP1;2.

dbSWEET: An Integrated Resource for SWEET Superfamily to Understand, Analyze and Predict the Function of Sugar Transporters in Prokaryotes and Eukaryotes.

SWEET (Sweet Will Eventually be Exported Transporter) proteins have been recently discovered and form one of the three major families of sugar transporters. Homologs of SWEET are found in both prokaryotes and eukaryotes. Bacterial SWEET homologs have three transmembrane segments forming a triple-helical bundle and the functional form is dimers. Eukaryotic SWEETs have seven transmembrane helical segments forming two triple-helical bundles with a linker helix. Members of SWEET homologs have been shown to be involved in several important physiological processes in plants. However, not much is known regarding the biological significance of SWEET homologs in prokaryotes and in mammals. We have collected more than 2000 SWEET homologs from both prokaryotes and eukaryotes. For each homolog, we have modeled three different conformational states representing outward open, inward open and occluded states. We have provided details regarding substrate-interacting residues and residues forming the selectivity filter for each SWEET homolog. Several search and analysis options are available. The users can generate a phylogenetic tree and structure-based sequence alignment for selected set of sequences. With no metazoan SWEETs functionally characterized, the features observed in the selectivity filter residues can be used to predict the potential substrates that are likely to be transported across the metazoan SWEETs. We believe that this database will help the researchers to design mutational experiments and simulation studies that will aid to advance our understanding of the physiological role of SWEET homologs. This database is freely available to the scientific community at http://bioinfo.iitk.ac.in/bioinfo/dbSWEET/Home.

Imidazole Nitrogens of Two Histidine Residues Participating in N-H···N Hydrogen Bonds in Protein Structures: Structural Bioinformatics Approach Combined with Quantum Chemical Calculations.

Protein structures are stabilized by different types of hydrogen bonds. However, unlike the DNA double helical structure, the N-H···N type of hydrogen bonds is relatively rare in proteins. N-H···N hydrogen bonds formed by imidazole groups of two histidine residues have not been investigated. We have systematically analyzed 5333 high-resolution protein structures with resolution 1.8 Å or better and identified 285 histidine pairs in which the nitrogen atoms of the imidazole side chains can potentially participate in N-H···N hydrogen bonds. The histidine pairs were further divided into two groups, neutral-neutral and protonated-neutral, depending on the protonation state of the donor histidine. Quantum chemical calculations were performed on imidazole groups adopting the same geometry observed in the protein structures. Average interaction energies between the interacting imidazole groups are -6.45 and -22.5 kcal/mol for neutral-neutral and protonated-neutral, respectively. Hydrogen bond interaction between the imidazole moieties is further confirmed by natural bond orbital analyses of the model compounds. Histidine residues involved in N-H···N hydrogen bonds are relatively more buried and have low B-factor values in the protein structures. N-H···N hydrogen bond formed by a pair of buried histidine residues can significantly contribute to the structural stability of proteins.

Anion-selective Formate/nitrite transporters: taxonomic distribution, phylogenetic analysis and subfamily-specific conservation pattern in prokaryotes.

BACKGROUND: The monovalent anions formate, nitrite and hydrosulphide are main metabolites of bacterial respiration during anaerobic mixed-acid fermentation. When accumulated in the cytoplasm, these anions become cytotoxic. Membrane proteins that selectively transport these monovalent anions across the membrane have been identified and they belong to the family of Formate/Nitrite Transporters (FNTs). Individual members that selectively transport formate, nitrite and hydrosulphide have been investigated. Experimentally determined structures of FNTs indicate that they share the same hourglass helical fold with aquaporins and aquaglyceroporins and have two constriction regions, namely, cytoplasmic slit and central constriction. Members of FNTs are found in bacteria, archaea, fungi and protists. However, no FNT homolog has been identified in mammals. With FNTs as potential drug targets for many bacterial diseases, it is important to understand the mechanism of selectivity and transport across these transporters. RESULTS: We have systematically searched the sequence databases and identified 2206 FNT sequences from bacteria, archaea and eukaryotes. Although FNT sequences are very diverse, homology modeling followed by structure-based sequence alignment revealed that nearly one third of all the positions within the transmembrane region exhibit high conservation either as a group or at the level of individual residues across all three kingdoms. Phylogenetic analysis of prokaryotic FNT sequences revealed eight different subgroups. Formate, nitrite and hydrosulphide transporters respectively are clustered into two (FocA and FdhC), three (NirC-α, NirC-ß and NirC-γ) and one (HSC) subfamilies. We have also recognized two FNT subgroups (YfdC-α and YfdC-ß) with unassigned function. Analysis of taxonomic distribution indicates that each subfamily prefers specific taxonomic groups. Structure-based sequence alignment of individual subfamily members revealed that certain positions in the two constriction regions and some residues facing the interior show subfamily-specific conservation. We have also identified examples of FNTs with the two constriction regions formed by residues that are less frequently observed. We have developed dbFNT, a database of FNT models and associated details. dbFNT is freely available to scientific community. CONCLUSIONS: Taxonomic distribution and sequence conservation of FNTs exhibit subfamily-specific features. The conservation pattern in the central constriction and cytoplasmic slit in the open and closed states are distinct for YfdC and NirC subfamilies. The same is true for some residues facing the interior of the transporters. The specific residues in these positions can exert influence on the type of solutes that are transported by these proteins. With FNTs found in many disease-causing bacteria, the knowledge gained in this study can be used in the development and design of anti-bacterial drugs.

Comparison of metal-binding strength between methionine and cysteine residues: Implications for the design of metal-binding motifs in proteins.

Metals play vital role in various physiological processes and are bound to biomolecules. Although cysteine sulfur is more frequently found as metal-binding ligand, methionine prefers to occur in copper-binding motifs of some proteins. To address methionine's lower preference in copper-binding sites in comparison to cysteine, we have considered copper-binding motifs (His-Cys-His-Met) from seven different high-resolution protein structures. We performed quantum chemical calculations to find out the strength of interactions between sulfur and metal ion in both Met and Cys residues. In the case of Cys, both neutral (CysH) and the deprotonated form (Cys-) were considered. We used two different levels of theory (B3LYP and M06-2X) and the model compounds methyl propyl sulfide, ethanethiol and ethanethiolate were used to represent Met, CysH and Cys- respectively. To compare the metal-binding strength, we mutated Met in silico to CysH/Cys- and performed the calculations. We also carried out calculations with wild-type Cys present in the same metal-binding motif. On average, interactions of Met with copper ion are stronger by 13-35kcal/mol compared to CysH. However, Cys- interactions with copper is stronger than that of Met by ~250kcal/mol. We then considered the entire metal-binding motif with four residues and calculated the interaction energies with the copper ion. We also considered Met→Cys- mutation in the motif and repeated the calculations. Interaction of the wild-type motif with the copper ion is ~160kcal/mol weaker than that of mutated motif. Our studies suggest the factors that could explain why Met is not as frequently observed as Cys in the metal-binding motifs. Results of these studies will help in designing metal-binding motifs in proteins with varying interaction strengths.

Binding affinity of pro-apoptotic BH3 peptides for the anti-apoptotic Mcl-1 and A1 proteins: Molecular dynamics simulations of Mcl-1 and A1 in complex with six different BH3 peptides.

The anti-apoptotic members of Bcl-2 family of proteins bind to their pro-apoptotic counterparts to induce or prevent cell death.Based on the distinct binding profiles for specific pro-apoptotic BH3 peptides, the anti-apoptotic Bcl-2 proteins can be divided into at least two subclasses. The subclass that includes Bcl-XL binds strongly to Bad BH3 peptide while it has weak binding affinity for the second subclass of Bcl-2 proteins such as Mcl-1 and A1. Anti-apoptotic Bcl-2 proteins are considered to be attractive drug targets for anti-cancer drugs. BH3-mimetic inhibitors such as ABT-737 have been shown to be specific to Bcl-XL subclass while Mcl-1 and A1 show resistance to the same drug. An efficacious inhibitor should target all the anti-apoptotic Bcl-2 proteins. Hence, development of inhibitors selective to Mcl-1 and A1 is of prime importance for targeted cancer therapeutics. The first step to achieve this goal is to understand the molecular basis of high binding affinities of specific pro-apoptotic BH3 peptides for Mcl-1 and A1. To understand the interactions between the BH3 peptides and Mcl-1/A1, we performed multi-nanosecond molecular dynamics (MD) simulations of six complex structures of Mcl-1 and A1. With the exception of Bad, all complex structures were experimentally determined. Bad complex structures were modeled. Our simulation studies identified specific pattern of polar interactions between Mcl-1/A1 and high-affinity binding BH3 peptides. The lack of such polar interactions in Bad peptide complex is attributed to specific basic residues present before and after the highly conserved Leu residue. The close approach of basic residues in Bad and Mcl-1/A1 is hypothesized to be the cause of weak binding affinity. To test this hypothesis, we generated in silico mutants of these basic residues in Bad peptide and Mcl-1/A1 proteins. MD simulations of the mutant systems established the pattern of stable polar interactions observed in high-affinity binding BH3 peptides. We have thus identified specific residue positions in Bad and Mcl-1/A1 responsible for the weak binding affinity. Results from these simulation studies will aid in the development of inhibitors specific to Mcl-1 and A1 proteins.

N-H···N Hydrogen Bonds Involving Histidine Imidazole Nitrogen Atoms: A New Structural Role for Histidine Residues in Proteins.

The amino acid histidine can play a significant role in the structure and function of proteins. Its various functions include enzyme catalysis, metal binding activity, and involvement in cation-π, π-π, salt-bridge, and other types of noncovalent interactions. Although histidine's imidazole nitrogens (Nδ and Nε) are known to participate in hydrogen bond (HB) interactions as an acceptor or a donor, a systematic study of N-H···N HBs with the Nδ/Nε atom as the acceptor has not been conducted. In this study, we have examined two data sets of ultra-high-resolution (data set I) and very high-resolution (data set II) protein structures and identified 28 and 4017 examples of HBs of the N-H···Nδ/Nε type from both data sets involving histidine imidazole nitrogen as the acceptor. In nearly 70% of them, the main-chain N-H bond is the HB donor, and a majority of the examples are from the N-H group separated by two residues (Ni+2-Hi+2) from histidine. Quantum chemical calculations using model compounds were performed with imidazole and N-methylacetamide, and they assumed conformations from 19 examples from data set I with N-H···Nδ/Nε HBs. Basis set superposition error-corrected interaction energies varied from -5.0 to -6.78 kcal/mol. We also found that the imidazole nitrogen of 9% of histidine residues forming N-H···Nδ/Nε interactions in data set II participate in bifurcated HBs. Natural bond orbital analyses of model compounds indicate that the strength of each HB is mutually influenced by the other. Histidine residues involved in Ni+2-Hi+2···Nδi/Nεi HBs are frequently observed in a specific N-terminal capping position giving rise to a novel helix-capping motif. Along with their predominant occurrence in loop segments, we propose a new structural role for histidines in protein structures.

Unconventional N-H N Hydrogen Bonds Involving Proline Backbone Nitrogen in Protein Structures.

Contrary to DNA double-helical structures, hydrogen bonds (H-bonds) involving nitrogen as the acceptor are not common in protein structures. We systematically searched N-H…N H-bonds in two different sets of protein structures. Data set I consists of neutron diffraction and ultrahigh-resolution x-ray structures (0.9 Å resolution or better) and the hydrogen atom positions in these structures were determined experimentally. Data set II contains structures determined using x-ray diffraction (resolution ≤ 1.8 Å) and the positions of hydrogen atoms were generated using a computational method. We identified 114 and 14,347 potential N-H…N H-bonds from these two data sets, respectively, and 56-66% of these were of the Ni+1-Hi+1…Ni type, with Ni being the proline backbone nitrogen. To further understand the nature of such unusual contacts, we performed quantum chemical calculations on the model compound N-acetyl-L-proline-N-methylamide (Ace-Pro-NMe) with coordinates taken from the experimentally determined structures. A potential energy profile generated by varying the ψ dihedral angle in Ace-Pro-NMe indicates that the conformation with the N-H…N H-bond is the most stable. An analysis of H-bond-forming proline residues reveals that more than 30% of the proline carbonyl groups are also involved in n → π(∗) interactions with the carbonyl carbon of the preceding residue. Natural bond orbital analyses demonstrate that the strength of N-H…N H-bonds is less than half of that observed for a conventional H-bond. This study clearly establishes the H-bonding capability of proline nitrogen and its prevalence in protein structures. We found many proteins with multiple instances of H-bond-forming prolines. With more than 15% of all proline residues participating in N-H…N H-bonds, we suggest a new, to our knowledge, structural role for proline in providing stability to loops and capping regions of secondary structures in proteins.

Controlling in Vitro Insulin Amyloidosis with Stable Peptide Conjugates: A Combined Experimental and Computational Study.

Insulin aggregation, to afford amyloidogenic polypeptide fibrils, is an energetically driven, well-studied phenomenon, which presents interesting biological ramifications. These aggregates are also known to form around insulin injection sites and in diabetic patients suffering from Parkinson's disease. Such occurrences force considerable reduction in hormone activity and are often responsible for necrotic deposits in diabetic patients. Changes in physicochemical environment, such as pH, temperature, ionic strength, and mechanical agitation, affect insulin fibrillation, which also presents intrigue from the structural viewpoint. Several reports have tried to unravel underlying mechanisms concerning the aggregation process taking into account a three aromatic amino acid patch Phe(B24)-Phe(B25)-Tyr(B26) located in the C-terminal part of the B chain, identified as a key site for human insulin-receptor interaction. The present study describes design and inhibitory effects of novel peptide conjugates toward fibrillation of insulin as investigated by thioflavin T assay, circular dichroism, and AFM. Possible interaction of insulin with peptide-based fibrillation inhibitors reveals an important role of hydrophobic interactions in the inhibition process. Molecular dynamics simulation studies demonstrate that inhibitor D4 interacts with insulin residues from the helix and the C-terminal extended segment of chain B. These studies present a novel approach for the discovery of stable, peptide-based ligands as novel antiamyloidogenic agents for insulin aggregation.

Major intrinsic protein superfamily: channels with unique structural features and diverse selectivity filters.

Members of the superfamily of major intrinsic proteins (MIPs) facilitate water and solute permeability across cell membranes and are found in sources ranging from bacteria to humans. Aquaporin and aquaglyceroporin channels are the prominent members of the MIP superfamily. Experimental studies show that MIPs are involved in important physiological processes in mammals and plants. They are implicated in several human diseases and are considered to be attractive drug targets for a wide range of diseases such as cancer, brain edema, epilepsy, glaucoma, and congestive heart failure. Three-dimensional structures of MIP channels from diverse sources reveal that MIPs adopt a unique conserved hourglass helical fold consisting of six transmembrane helices (TM1-TM6) and two half-helices (LB and LE). Conserved NPA motifs near the center and the aromatic/arginine selectivity filter (Ar/R SF) toward the extracellular side constitute two narrow constriction regions within the channel. Structural knowledge combined with simulation studies have helped to investigate the role of these two constriction regions in the transport and selectivity of the solutes. With the availability of many genome sequences from diverse species, a large number of MIP genes have been identified. Homology models of 1500 MIP channels have been used to derive structure-based sequence alignment of TM1-TM6 helices and the two half-helices LB and LE. Thirteen residues are highly conserved in different transmembrane helices and half-helices. High group conservation of small and weakly polar residues is observed in 27 positions at the interface of two interacting helices. Thus, although the MIP sequences are diverse, the hourglass helical fold is maintained during evolution with the conservation of these 40 positions within the transmembrane region. We have proposed a generic structure-based numbering scheme for the MIP channels that will facilitate easier comparison of the MIP sequences. Analysis of Ar/R SF in all 1500 MIPs indicates the extent of diversity in the four residues that form this narrow region. Certain residues are completely avoided in the SF, even if they have the same chemical nature as that of the most frequently observed residues. For example, arginine is the most preferred residue in a specific position of Ar/R SF, whereas lysine is almost always avoided in any of the four positions. MIP channels with highly hydrophobic or hydrophilic Ar/R SF have been identified. Similarly, there are examples of MIP channels in which all four residues of Ar/R SF are bulky, thus almost occluding the pore. Many plant MIPs possess small residues at all SF positions, resulting in a larger pore diameter. A majority of MIP channels are yet to be functionally characterized, and their in vivo substrates are not yet identified. A complete understanding of the relationship between the nature of Ar/R SF and the solutes that are transported is required to exploit MIP channels as potential drug targets.

Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins.

The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10-15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport.