Rational design of a highly immunogenic prefusion-stabilized F glycoprotein antigen for a respiratory syncytial virus vaccine.

Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.

Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions.

During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.

MntC-Dependent Manganese Transport Is Essential for Staphylococcus aureus Oxidative Stress Resistance and Virulence.

Staphylococcus aureus is a human pathogen that has developed several approaches to evade the immune system, including a strategy to resist oxidative killing by phagocytes. This resistance is mediated by production of superoxide dismutase (SOD) enzymes which use manganese as a cofactor. S. aureus encodes two manganese ion transporters, MntABC and MntH, and a possible Nramp family manganese transporter, exemplified by S. aureus N315 SA1432. Their relative contributions to manganese transport have not been well defined in clinically relevant isolates. For this purpose, insertional inactivation mutations were introduced into mntC, mntH, and SA1432 individually and in combination. mntC was necessary for full resistance to methyl viologen, a compound that generates intracellular free radicals. In contrast, strains with an intact mntH gene had a minimal increase in resistance that was revealed only in mntC strains, and no change was observed upon mutation of SA1432 in strains lacking both mntC and mntH Similarly, MntC alone was required for high cellular SOD activity. In addition, mntC strains were attenuated in a murine sepsis model. To further link these observations to manganese transport, an S. aureus MntC protein lacking manganese binding activity was designed, expressed, and purified. While circular dichroism experiments demonstrated that the secondary and tertiary structures of this protein were unaltered, a defect in manganese binding was confirmed by isothermal titration calorimetry. Unlike complementation with wild-type mntC, introduction of the manganese-binding defective allele into the chromosome of an mntC strain did not restore resistance to oxidative stress or virulence. Collectively, these results underscore the importance of MntC-dependent manganese transport in S. aureus oxidative stress resistance and virulence.IMPORTANCE Work outlined in this report demonstrated that MntC-dependent manganese transport is required for S. aureus virulence. These study results support the model that MntC-specific antibodies elicited by a vaccine have the potential to disrupt S. aureus manganese transport and thus abrogate to its virulence.

High Resolution Mapping of Bactericidal Monoclonal Antibody Binding Epitopes on Staphylococcus aureus Antigen MntC.

The Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups (mAB 305-72-5 - group 1, mAB 305-78-7 - group 2, and mAB 305-101-8 - group 3) were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS). All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7) with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity.

The N-terminal capping propensities of the D-helix modulate the allosteric activation of the Escherichia coli cAMP receptor protein.

Transduction of biological signals at the molecular level involves the activation and/or inhibition of allosteric proteins. In the transcription factor cAMP receptor protein (CRP) from Escherichia coli, the allosteric activation, or apo-holo transition, involves rigid body motions of domains and structural rearrangements within the hinge region connecting the cAMP- and DNA-binding domains. During this apo-holo transition, residue 138 is converted as part of the elongated D-helix to the position of the N-terminal capping residue of a shorter D-helix. The goal of the current study is to elucidate the role of residue 138 in modulating the allostery between cAMP and DNA binding. By systematically mutating residue 138, we found that mutants with higher N-terminal capping propensities lead to increased cooperativity of cAMP binding and a concomitant increase in affinity for lac-DNA. Furthermore, mutants with higher N-terminal capping propensity correlate with properties characteristic of holo-CRP, particularly, increase in protein structural dynamics. Overall, our results provide a quantitative characterization of the role of residue 138 in the isomerization equilibrium between the apo and holo forms of CRP, and in turn the thermodynamic underpin to the molecular model of allostery revealed by the high resolution structural studies.

Pb2+ as modulator of protein-membrane interactions.

Lead is a potent environmental toxin that mimics the effects of divalent metal ions, such as zinc and calcium, in the context of specific molecular targets and signaling processes. The molecular mechanism of lead toxicity remains poorly understood. The objective of this work was to characterize the effect of Pb(2+) on the structure and membrane-binding properties of C2α. C2α is a peripheral membrane-binding domain of Protein Kinase Cα (PKCα), which is a well-documented molecular target of lead. Using NMR and isothermal titration calorimetry (ITC) techniques, we established that C2α binds Pb(2+) with higher affinity than its natural cofactor, Ca(2+). To gain insight into the coordination geometry of protein-bound Pb(2+), we determined the crystal structures of apo and Pb(2+)-bound C2α at 1.9 and 1.5 Å resolution, respectively. A comparison of these structures revealed that the metal-binding site is not preorganized and that rotation of the oxygen-donating side chains is required for the metal coordination to occur. Remarkably, we found that holodirected and hemidirected coordination geometries for the two Pb(2+) ions coexist within a single protein molecule. Using protein-to-membrane Förster resonance energy transfer (FRET) spectroscopy, we demonstrated that Pb(2+) displaces Ca(2+) from C2α in the presence of lipid membranes through the high-affinity interaction with the membrane-unbound C2α. In addition, Pb(2+) associates with phosphatidylserine-containing membranes and thereby competes with C2α for the membrane-binding sites. This process can contribute to the inhibitory effect of Pb(2+) on the PKCα activity.

Exchange protein directly activated by cyclic AMP isoform 2 is not a direct target of sulfonylurea drugs.

It has been reported by Zhang et al. that antidiabetic sulfonylurea drugs promote insulin secretion by directly binding to exchange protein directly activated by cyclic AMP isoform 2 (Epac2) and activating its down-stream effector Rap1. However, a critical link for an unambiguous validation of a direct interaction between Epac2 and sulfonylurea using purified individual components is missing. Our in vitro analyses using purified full-length Epac2 and Rap1 suggest that sulfonylureas are not able to directly bind to Epac2, nor are they capable of triggering Epac2-dependent Rap1 activation.

Rational stabilization of enzymes by computational redesign of surface charge-charge interactions.

Here, we report the application of a computational approach that allows the rational design of enzymes with enhanced thermostability while retaining full enzymatic activity. The approach is based on the optimization of the energy of charge-charge interactions on the protein surface. We experimentally tested the validity of the approach on 2 human enzymes, acylphosphatase (AcPh) and Cdc42 GTPase, that differ in size (98 vs. 198-aa residues, respectively) and tertiary structure. We show that the designed proteins are significantly more stable than the corresponding WT proteins. The increase in stability is not accompanied by significant changes in structure, oligomerization state, or, most importantly, activity of the designed AcPh or Cdc42. This success of the design methodology suggests that it can be universally applied to other enzymes, on its own or in combination with the other strategies based on redesign of the interactions in the protein core.

Cooperativity of complex salt bridges.

The energetic contribution of complex salt bridges, in which one charged residue (anchor residue) forms salt bridges with two or more residues simultaneously, has been suggested to have importance for protein stability. Detailed analysis of the net energetics of complex salt bridge formation using double- and triple-mutant cycle analysis revealed conflicting results. In two cases, it was shown that complex salt bridge formation is cooperative, i.e., the net strength of the complex salt bridge is more than the sum of the energies of individual pairs. In one case, it was reported that complex salt bridge formation is anti-cooperative. To resolve these different findings, we performed analysis of the geometries of salt bridges in a representative set of structures from the PDB and found that over 87% of all complex salt bridges anchored by Arg/Lys have a geometry such that the angle formed by their Calpha atoms, Theta, is <90 degrees . This preferred geometry is observed in the two reported instances when the energetics of complex salt bridge formation is cooperative, while in the reported anti-cooperative complex salt bridge, Theta is close to 160 degrees . Based on these observations, we hypothesized that complex salt bridges are cooperative for Theta < 90 degrees and anti-cooperative for 90 degrees < Theta < 180 degrees . To provide a further experimental test for this hypothesis, we engineered a complex salt bridge with Theta = 150 degrees into a model protein, the activation domain of human procarboxypeptidase A2 (ADA2h). Experimentally derived stabilities of the ADA2h variants allowed us to show that the complex salt bridge in ADA2h is anti-cooperative.

Role of the charge-charge interactions in defining stability and halophilicity of the CspB proteins.

Charge-charge interactions on the surface of native proteins are important for protein stability and can be computationally redesigned in a rational way to modulate protein stability. Such computational effort led to an engineered protein, CspB-TB that has the same core as the mesophilic cold shock protein CspB-Bs from Bacillus subtilis, but optimized distribution of charge-charge interactions on the surface. The CspB-TB protein shows an increase in the transition temperature by 20 degrees C relative to the unfolding temperature of CspB-Bs. The CspB-TB and CspB-Bs protein pair offers a unique opportunity to further explore the energetics of charge-charge interactions as the substitutions at the same sequence positions are done in largely similar structural but different electrostatic environments. In particular we addressed two questions. What is the contribution of charge-charge interactions in the unfolded state to the protein stability and how amino acid substitutions modulate the effect of increase in ionic strength on protein stability (i.e. protein halophilicity). To this end, we experimentally measured the stabilities of over 100 variants of CspB-TB and CspB-Bs proteins with substitutions at charged residues. We also performed computational modeling of these protein variants. Analysis of the experimental and computational data allowed us to conclude that the charge-charge interactions in the unfolded state of two model proteins CspB-Bs and CspB-TB are not very significant and computational models that are based only on the native state structure can adequately, i.e. qualitatively (stabilizing versus destabilizing) and semi-quantitatively (relative rank order), predict the effects of surface charge neutralization or reversal on protein stability. We also show that the effect of ionic strength on protein stability (protein halophilicity) appears to be mainly due to the screening of the long-range charge-charge interactions.

Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding.

The heat capacity change upon unfolding (deltaC(p)) is a thermodynamic parameter that defines the temperature dependence of the thermodynamic stability of proteins; however, physical basis of the heat capacity change is not completely understood. Although empirical surface area-based calculations can predict heat capacity changes reasonably well, accumulating evidence suggests that changes in hydration of those surfaces is not the only parameter contributing to the observed heat capacity changes upon unfolding. Because packing density in the protein interior is similar to that observed in organic crystals, we hypothesized that changes in protein dynamics resulting in increased rigidity of the protein structure might contribute to the observed heat capacity change upon unfolding. Using differential scanning calorimetry we characterized the thermodynamic behavior of a serine protease inhibitor eglin C and two eglin C variants with altered native state dynamics, as determined by NMR. We found no evidence of changes in deltaC(p) in either of the variants, suggesting that changes in rigidity do not contribute to the heat capacity change upon unfolding in this model system.

Protein stability and surface electrostatics: a charged relationship.

Engineering proteins to withstand a broad range of conditions continues to be a coveted objective, holding the potential to advance biomedicine, industry, and our understanding of disease. One way of achieving this goal lies in elucidating the underlying interactions that define protein stability. It has been shown that the hydrophobic effect, hydrogen bonding, and packing interactions between residues in the protein interior are dominant factors that define protein stability. The role of surface residues in protein stability has received much less attention. It has been believed that surface residues are not important for protein stability particularly because their interactions with the solvent should be similar in the native and unfolded states. In the case of surface charged residues, it was sometimes argued that solvent exposure meant that the high dielectric of the solvent will further decrease the strength of the charge-charge interactions. In this paper, we challenge the notion that the surface charged residues are not important for protein stability. We computationally redesigned sequences of five different proteins to optimize the surface charge-charge interactions. All redesigned proteins exhibited a significant increase in stability relative to their parent proteins, as experimentally determined by circular dichroism spectroscopy and differential scanning calorimetry. These results suggest that surface charge-charge interactions are important for protein stability and that rational optimization of charge-charge interactions on the protein surface can be a viable strategy for enhancing protein stability.

Dissecting the energetics of protein alpha-helix C-cap termination through chemical protein synthesis.

The alpha-helix is a fundamental protein structural motif and is frequently terminated by a glycine residue. Explanations for the predominance of glycine at the C-cap terminal portions of alpha-helices have invoked uniquely favorable energetics of this residue in a left-handed conformation or enhanced solvation of the peptide backbone because of the absence of a side chain. Attempts to quantify the contributions of these two effects have been made previously, but the issue remains unresolved. Here we have used chemical protein synthesis to dissect the energetic basis of alpha-helix termination by comparing a series of ubiquitin variants containing an L-amino acid or the corresponding D-amino acid at the C-cap Gly35 position. D-Amino acids can adopt a left-handed conformation without energetic penalty, so the contributions of conformational strain and backbone solvation can thus be separated. Analysis of the thermodynamic data revealed that the preference for glycine at the C' position of a helix is predominantly a conformational effect.

Mechanism of thermostabilization in a designed cold shock protein with optimized surface electrostatic interactions.

Using computational and sequence analysis of bacterial cold shock proteins, we designed a protein (CspB-TB) that has the core residues of mesophilic protein from Bacillus subtilis(CspB-Bs) and altered distribution of surface charged residues. This designed protein was characterized by circular dichroism spectroscopy, and found to have secondary and tertiary structure similar to that of CspB-Bs. The activity of the CspB-TB protein as measured by the affinity to a single-stranded DNA (ssDNA) template at 25 degrees C is somewhat higher than that of CspB-Bs. Furthermore, the decrease in the apparent binding constant to ssDNA upon increase in temperature is much more pronounced for CspB-Bs than for CspB-TB. Temperature-induced unfolding (as monitored by differential scanning calorimetry and circular dichroism spectroscopy) and urea-induced unfolding experiments were used to compare the stabilities of CspB-Bs and CspB-TB. It was found that CspB-TB is approximately 20 degrees C more thermostable than CspB-Bs. The thermostabilization of CspB-TB relative to CspB-Bs is achieved by decrease in the enthalpy and entropy of unfolding without affecting their temperature dependencies, i.e. these proteins have similar heat capacity changes upon unfolding. These changes in the thermodynamic parameters result in the global stability function, i.e. Gibbs energy, deltaG(T), that is shifted to higher temperatures with only small changes in the maximum stability. Such a mechanism of thermostabilization, although predicted from the basic thermodynamic considerations, has never been identified experimentally.

Conformational and thermodynamic properties of peptide binding to the human S100P protein.

S100P is a member of the S100 subfamily of calcium-binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target-recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca2+-dependent and -independent manner. Ca2+-independent binding occurs with low affinity (Kd approximately 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca2+-dependent binding of melittin to S100P occurs with high affinity (Kd approximately 5 microM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+-dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+-S100P. These results support a model for the Ca2+-dependent conformational switch in S100P for functional target recognition.