On the Structure of the Proton-Binding Site in the Fo Rotor of Chloroplast ATP Synthases

The recently reported crystal structures of the membrane-embedded proton-dependent c-ring rotors of a cyanobacterial F₁F_o ATP synthase and a chloroplast F₁F_o ATP synthase have provided new insights into the mechanism of this essential enzyme. While the overall features of these c-rings are similar, a discrepancy in the structure and hydrogen-bonding interaction network of the H⁺ sites suggests two distinct binding modes, potentially reflecting a mechanistic differentiation. Importantly, the conformation of the key glutamate side chain to which the proton binds is also altered. To investigate the nature of these differences, we use molecular dynamics simulations of both c-rings embedded in a phospholipid membrane. We observe that the structure of the c₁₅ ring from Spirulina platensis is unequivocally stable within the simulation time. By contrast, the proposed structure of the H⁺ site in the chloroplast c₁₄ ring changes rapidly and consistently into that reported for the c₁₅ ring, indicating that the latter represents a common binding mode. To assess this hypothesis, we have remodeled the c₁₄ ring by molecular replacement using the published structure factors. The resulting structure provides clear evidence in support of a common binding site conformation and is also considerably improved statistically. These findings, taken together with a sequence analysis of c-subunits in the ATP synthase family, indicate that the so-called proton-locked conformation observed in the c₁₅ ring may be a common characteristic not only of light-driven systems such as chloroplasts and cyanobacteria but also of a selection of other bacterial species.     

Systematic Mutational Analysis of Peptide Inhibition of the p53-MDM2/MDMX Interactions

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of ^17-28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or ^17-28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and ^17-28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and ^17-28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective K_d values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-^25-109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.     

Dynamics may significantly influence the estimation of interatomic distances in biomolecular X-ray structures

Atomic positions obtained by X-ray crystallography are time and space averages over many molecules in the crystal. Importantly, interatomic distances, calculated between such average positions and frequently used in structural and mechanistic analyses, can be substantially different from the more appropriate time-average and ensemble-average interatomic distances. Using crystallographic B-factors, one can deduce corrections, which have so far been applied exclusively to small molecules, to obtain correct average distances as a function of the type of atomic motion. Here, using 4774 high-quality protein X-ray structures, we study the significance of such corrections for different types of atomic motion. Importantly, we show that for distances shorter than 5 Å, corrections greater than 0.5 Å may apply, especially for noncorrelated or anticorrelated motion. For example, 14% of the studied structures have at least one pair of atoms with a correction of ≥ 0.5 Å in the case of noncorrelated motion. Using molecular dynamics simulations of villin headpiece, ubiquitin, and SH3 domain unit cells, we demonstrate that the majority of average interatomic distances in these proteins agree with noncorrelated corrections, suggesting that such deviations may be truly relevant. Importantly, we demonstrate that the corrections do not significantly affect stereochemistry and the overall quality of final refined X-ray structures, but can provide marked improvements in starting unrefined models obtained from low-resolution X-ray data. Finally, we illustrate the potential mechanistic and biological significance of the calculated corrections for KcsA ion channel and show that they provide indirect evidence that motions in its selectivity filter are highly correlated.     

Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations

Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P_i, ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the γ-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.     

Structural and kinetic analysis of pyrrolidine-based inhibitors of the drug-resistant Ile84Val mutant of HIV-1 protease

Human immunodeficiency virus (HIV) protease is a well-established drug target in HIV chemotherapy. However, continuously increasing resistance towards approved drugs inevitably requires the development of new inhibitors preferably showing no susceptibility against resistant HIV protease strains. Recently, symmetric pyrrolidine-3,4-bis-N-benzyl-sulfonamides have been developed as a new class of HIV-1 protease inhibitors. The most promising candidate exhibited a K_i of 74 nM towards a wild-type protease. Herein, we report the influence of the active-site mutations Ile50Val and Ile84Val on these inhibitors by structural and kinetic analysis. Although the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the important Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases, we were able to elucidate the structural basis of this phenomenon.     

Dynamics, Energetics, and Selectivity of the Low-K KcsA Channel Structure

Potassium channels are a diverse family of integral membrane proteins through which K⁺ can pass selectively. There is ongoing debate about the nature of conformational changes associated with the opening/closing and conductive/nonconductive states of potassium channels. The channels partly exert their function by varying their conductance through a mechanism known as C-type inactivation. Shortly after the activation of K⁺ channels, their selectivity filter stops conducting ions at a rate that depends on various stimuli. The molecular mechanism of C-type inactivation has not been fully understood yet. However, the X-ray structure of the KcsA channel obtained in the presence of low K⁺ concentration is thought to be representative of a K⁺ channel in the C-type inactivated state. Here, extensive, fully atomistic molecular dynamics and free-energy simulations of the low-K⁺ KcsA structure in an explicit lipid bilayer are performed to evaluate the stability of this structure and the selectivity of its binding sites. We find that the low-K⁺ KcsA structure is stable on the timescale of the molecular dynamics simulations performed, and that ions preferably remain in S1 and S4. In the absence of ions, the selectivity filter evolves toward an asymmetric architecture, as already observed in other computations of the high-K⁺ structure of KcsA and KirBac. The low-K⁺ KcsA structure is not permeable by Na⁺, K⁺, or Rb⁺, and the selectivity of its binding sites is different from that of the high-K⁺ structure.     

Statics of the Ribosomal Exit Tunnel: Implications for Cotranslational Peptide Folding, Elongation Regulation, and Antibiotics Binding

A sophisticated interplay between the static properties of the ribosomal exit tunnel and its functional role in cotranslational processes is revealed by constraint counting on topological network representations of large ribosomal subunits from four different organisms. As for the global flexibility characteristics of the subunit, the results demonstrate a conserved stable structural environment of the tunnel. The findings render unlikely that deformations of the tunnel move peptides down the tunnel in an active manner. Furthermore, the stable environment rules out that the tunnel can adapt widely so as to allow tertiary folding of nascent chains. Nevertheless, there are local zones of flexible nucleotides within the tunnel, between the peptidyl transferase center and the tunnel constriction, and at the tunnel exit. These flexible zones strikingly agree with previously identified folding zones. As for cotranslational elongation regulation, flexible residues in the β-hairpin of the ribosomal L22 protein were verified, as suggested previously based on structural results. These results support the hypothesis that L22 can undergo conformational changes that regulate the tunnel voyage of nascent polypeptides. Furthermore, rRNA elements, for which conformational changes have been observed upon interaction of the tunnel wall with a nascent SecM peptide, are less strongly coupled to the subunit core. Sequences of coupled rigid clusters are identified between the tunnel and some of these elements, suggesting signal transmission by a domino-like mechanical coupling. Finally, differences in the flexibility of the glycosidic bonds of bases that form antibiotics-binding crevices within the peptidyl transferase center and the tunnel region are revealed for ribosomal structures from different kingdoms. In order to explain antibiotics selectivity, action, and resistance, according to these results, differences in the degrees of freedom of the binding regions may need to be considered.     

The recognition specificity of the CHD1 chromodomain with modified histone H3 peptides

Histone tail peptides comprise the flexible portion of chromatin, the substance which serves as the packaging for the eukaryotic genome. According to the histone code hypothesis, reader protein domains (chromodomains) can recognize modifications of amino acid residues within these peptides, regulating the expression of genes. We have performed simulations on models of chromodomain helicase DNA-binding protein 1 complexed with a variety of histone H3 modifications. Binding free energies for both the overall complexes and the individual residues within the protein and peptides were computed with molecular mechanics-generalized Born surface area. The simulation results agree well with experimental data and identify several chromodomain helicase DNA-binding protein 1 residues that play key roles in the interaction with each of the H3 modifications. We identified one class of protein residues that bind to H3 in all of the complexes (generally interacting hydrophobically), and a second class of residues that bind only to particular H3 modifications (generally interacting electrostatically). Additionally, we found that modifications of H3R2 and H3T3 have a dominant effect on the binding affinity; methylation of H3K4 has little effect on the interaction strength when H3R2 or H3T3 is modified. Our findings with regard to the specificity shown by the latter class of protein residues in their binding affinity to certain modifications of H3 support the histone code hypothesis.     

The interaction of cofilin with the actin filament

Cofilin is a key actin-binding protein that is critical for controlling the assembly of actin within the cell. Here, we present the results of molecular docking and dynamics studies using a muscle actin filament and human cofilin I. Guided by extensive mutagenesis results and other biophysical and structural studies, we arrive at a model for cofilin bound to the actin filament. This predicted structure agrees very well with electron microscopy results for cofilin-decorated filaments, provides molecular insight into how the known F- and G-actin sites on cofilin interact with the filament, and also suggests new interaction sites that may play a role in cofilin binding. The resulting atomic-scale model also helps us understand the molecular function and regulation of cofilin and provides testable data for future experimental and simulation work.     

Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

Human immunodeficiency virus type 1 (HIV-1) protease (PR) permits viral maturation by processing the gag and gag-pro-pol polyproteins. HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy but the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates consideration of drug resistance in novel drug design. Drug-resistant HIV-1 PR variants no longer inhibited efficiently, continue to hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we termed the substrate envelope. Earlier, we showed that resistance mutations arise where PIs protrude beyond the substrate envelope, because these regions are crucial for drug binding but not for substrate recognition. We extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. We simulated the molecular dynamics of seven PR-substrate complexes to estimate the conformational flexibility of the bound substrates. Interdependence of substrate-protease interactions might compensate for variations in cleavage-site sequences and explain how a diverse set of sequences are recognized as substrates by the same enzyme. This diversity might be essential for regulating sequential processing of substrates. We define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance.     

Structural and Thermodynamic Characterization of Pre- and Postpolymerization States in the F4 Fimbrial Subunit FaeG

Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27  versus ∼ 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.     

Ion Selectivity of the KcsA Channel: A Perspective from Multi-Ion Free Energy Landscapes

Potassium (K⁺) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K⁺ through cell membranes. Comprising five specific cation binding sites (S₀-S₄) formed by the backbone carbonyl groups of conserved residues common to all K⁺ channels, the narrow selectivity filter allows fast conduction of K⁺ while being highly selective for K⁺ over Na⁺. To extend our knowledge of the microscopic mechanism underlying selectivity in K⁺ channels, we characterize the free energy landscapes governing the entry and translocation of a Na⁺ or a K⁺ from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K⁺ in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K⁺ or Na⁺ in binding site S₀ or S₁ are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na⁺ than for K⁺, implying that the kinetics of ion conduction is slower when a Na⁺ enters the pore. Third, the region corresponding to binding site S₂ near the center of the narrow pore emerges as the most selective for K⁺ over Na⁺. In particular, while there is a stable minimum for K⁺ in site S₂, Na⁺ faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S₂ in the selectivity filter of K⁺ channels.     

Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase

Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity.We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl₂ or MnCl₂. In the E122Q mutant, k_cat is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2.     

Glycine-Rich Loop of Mitochondrial Processing Peptidase α-Subunit Is Responsible for Substrate Recognition by a Mechanism Analogous to Mitochondrial Receptor Tom20

Tryptophan fluorescence measurements were used to characterize the local dynamics of the highly conserved glycine-rich loop (GRL) of the mitochondrial processing peptidase (MPP) α-subunit in the presence of the substrate precursor. Reporter tryptophan residue was introduced into the GRL of the yeast α-MPP (Y299W) or at a proximal site (Y303W). Time-resolved and steady-state fluorescence spectroscopy demonstrated that for Trp299, the primary contact with the yeast malate dehydrogenase precursor evokes a change of the local GRL mobility. Moreover, time-resolved measurements showed that a functionless α-MPP with a single-residue deletion in the loop (Y303W/ΔG292) is defective particularly in the primary contact with substrate. Thus, the GRL was proved to be part of a contact site of the enzyme specifically recognizing the substrate. Regarding the surface exposure and presence of the hydrophobic patches within the GRL, we proposed a functional analogy between the presequence recognition by the hydrophobic binding groove of the Tom20 mitochondrial import receptor and the GRL of the α-MPP. A molecular dynamics (MD) simulation of the MPP-substrate peptide complex model was employed to test this hypothesis. The initial positioning and conformation of the substrate peptide in the model fitting were chosen based on the analogy of its interaction with the Tom20 binding groove. MD simulation confirmed the stability of the proposed interaction and showed also a decrease in GRL flexibility in the presence of substrate, in agreement with fluorescence measurements. Moreover, conserved substrate hydrophobic residues in positions + 1 and − 4 to the cleavage site remain in close contact with the side chains of the GRL during the entire production part of MD simulation as stabilizing points of the hydrophobic interaction. We conclude that the GRL of the MPP α-subunit is the crucial evolutional outcome of the presequence recognition by MPP and represents a functional parallel with Tom20 import receptor.     

Tracing the detail: how mutations affect binding modes and thermodynamic signatures of closely related aldose reductase inhibitors

Improvements on the computational methods for affinity prediction from the structure of protein-ligand complexes require a better understanding of the nature of molecular interactions and biomolecular recognition principles. In the present contribution, the binding of two chemically closely related human aldose reductase inhibitors had been studied by high-resolution X-ray analysis (0.92-1.35 ?) and isothermal titration calorimetry against a series of single-site mutants of the wild-type protein. A crucial threonine thought to be involved in a short bromine-to-oxygen halogen bond to the inhibitors in the wild type has been mutated to the structurally similar residues alanine, cysteine, serine and valine. Overall, structurally, the binding mode of the inhibitors is conserved; however, small but significant geometrical adaptations are observed as a consequence of the spatial and electronic changes at the mutation site. They involve the opening of a central bond angle and shifts in consequence of the lost or gained halogen bonds. Remarkably, the tiny structural changes are responded by partly strong modulation of the thermodynamic profiles. Even though the free energy of binding is maximally perturbed by only 7 kJ/mol, much stronger modulations and shifts in the enthalpy and entropy signatures are revealed, which indicate a pronounced enthalpy/entropy compensation. However, an explanatory correlation can be detected when facing these perturbances against the small structural changes. This also provides deeper insights into how single-site mutations can alter the selectivity profile of closely related ligands against a target protein.     

Backbone Flexibility of CDR3 and Immune Recognition of Antigens

Conformational entropy is an important component of protein–protein interactions; however, there is no reliable method for computing this parameter. We have developed a statistical measure of residual backbone entropy in folded proteins by using the ?–ψ distributions of the 20 amino acids in common secondary structures. The backbone entropy patterns of amino acids within helix, sheet or coil form clusters that recapitulate the branching and hydrogen bonding properties of the side chains in the secondary structure type. The same types of residues in coil and sheet have identical backbone entropies, while helix residues have much smaller conformational entropies. We estimated the backbone entropy change for immunoglobulin complementarity-determining regions (CDRs) from the crystal structures of 34 low-affinity T-cell receptors and 40 high-affinity Fabs as a result of the formation of protein complexes. Surprisingly, we discovered that the computed backbone entropy loss of only the CDR3, but not all CDRs, correlated significantly with the kinetic and affinity constants of the 74 selected complexes. Consequently, we propose a simple algorithm to introduce proline mutations that restrict the conformational flexibility of CDRs and enhance the kinetics and affinity of immunoglobulin interactions. Combining the proline mutations with rationally designed mutants from a previous study led to 2400-fold increase in the affinity of the A6 T-cell receptor for Tax-HLAA2. However, this mutational scheme failed to induce significant binding changes in the already-high-affinity C225–Fab/huEGFR interface. Our results will serve as a roadmap to formulate more effective target functions to design immune complexes with improved biological functions.     

Structural Basis for the Recognition and Cleavage of Polysialic Acid by the Bacteriophage K1F Tailspike Protein EndoNF

An α-2,8-linked polysialic acid (polySia) capsule confers immune tolerance to neuroinvasive, pathogenic prokaryotes such as Escherichia coli K1 and Neisseria meningitidis and supports host infection by means of molecular mimicry. Bacteriophages of the K1 family, infecting E. coli K1, specifically recognize and degrade this polySia capsule utilizing tailspike endosialidases. While the crystal structure for the catalytic domain of the endosialidase of bacteriophage K1F (endoNF) has been solved, there is yet no structural information on the mode of polySia binding and cleavage available. The crystal structure of activity deficient active-site mutants of the homotrimeric endoNF cocrystallized with oligomeric sialic acid identified three independent polySia binding sites in each endoNF monomer. The bound oligomeric sialic acid displays distinct conformations at each site. In the active site, a Sia₃ molecule is bound in an extended conformation representing the enzyme-product complex. Structural and biochemical data supported by molecular modeling enable to propose a reaction mechanism for polySia cleavage by endoNF.