Sublingual Priming with a HIV gp41-Based Subunit Vaccine Elicits Mucosal Antibodies and Persistent B Memory Responses in Non-Human Primates.

Persistent B cell responses in mucosal tissues are crucial to control infection against sexually transmitted pathogens like human immunodeficiency virus 1 (HIV-1). The genital tract is a major site of infection by HIV. Sublingual (SL) immunization in mice was previously shown to generate HIV-specific B cell immunity that disseminates to the genital tract. We report here the immunogenicity in female cynomolgus macaques of a SL vaccine based on a modified gp41 polypeptide coupled to the cholera toxin B subunit designed to expose hidden epitopes and to improve mucosal retention. Combined SL/intramuscular (IM) immunization with such mucoadhesive gp41-based vaccine elicited mucosal HIV-specific IgG and IgA antibodies more efficiently than IM immunization alone. This strategy increased the number and duration of gp41-specific IgA secreting cells. Importantly, combined immunization improved the generation of functional antibodies 3 months after vaccination as detected in HIV-neutralizing assays. Therefore, SL immunization represents a promising vaccine strategy to block HIV-1 transmission.

Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain.

The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.

Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti-HIV-1 drugs.

During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates.

Thermodynamic analysis of the binding of 2F5 (Fab and immunoglobulin G forms) to its gp41 epitope reveals a strong influence of the immunoglobulin Fc region on affinity.

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.

A comparative analysis of the folding and misfolding pathways of the third PDZ domain of PSD95 investigated under different pH conditions.

Equilibrium unfolding at neutral pH of the third PDZ domain of PSD95 is well described by the presence of a partly unfolded intermediate that presents association phenomena. After some days' incubation annular and fibrillar structures form from the oligomers. At pH values below 3, however, differential scanning calorimetry shows that PDZ3 seems to unfold under a two-state scheme. Kinetic measurements followed by dynamic light scattering, ThT and ANS fluorescence reveal that the misfolding pathway still exists despite the absence of any populated intermediates and shows an irreversible assembling of the supramacromolecular structures as well as an appreciable lag-phase, contrary to what is found in similar experiments at neutral pH. Moreover, as shown by transmission-electron-microscopy images, the annular structures seen at neutral pH completely disappear from incubated solutions. According to the structural information, this titration behavior appears to be the consequence of a conformational equilibrium that depends on the protonation of some Glu residues located at the C-terminal α3 helix and at the hairpin formed by strands ß2 and ß3. Our calculations suggest that the enthalpic contribution of these interactions may well be as much as 40kJ·mol(-1). The possible regulatory role of this equilibrium upon PDZ3 functionality and amyloid formation is briefly discussed.

An oligomeric equilibrium intermediate as the precursory nucleus of globular and fibrillar supramacromolecular assemblies in a PDZ domain.

The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60-70 degrees C small oligomers populate, showing a typical beta-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of approximately 12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of approximately 50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60 degrees C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ x mol(-1). Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions.

Thermodynamic characterization of the folding equilibrium of the human Nedd4-WW4 domain: at the frontiers of cooperative folding.

WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.

Structural and thermodynamic studies of Bergerac-SH3 chimeras.

Bergerac-type chimeras of spectrin SH3 were designed by extending a beta-hairpin by eight amino acids so that the extension protruded from the domain body like a "nose" being exposed to the solvent. A calorimetric study of several Bergerac-SH3 variants was carried out over a wide range of pH values and protein concentrations and the three-dimensional structure of one of them, SHH, was determined. X-ray studies confirmed that the nose had a well defined beta-structure whilst the chimera formed a stable tetramer within the crystal unit because of four tightly packed noses. In the pH range of 4-7 the heat-induced unfolding of some chimeras was complex and concentration dependent, whilst at pH values below 3.5, low protein concentrations of all the chimeras studied, including SHH, seemed to obey a monomolecular two-state unfolding model. The best set of data was obtained for the SHA variant, the unfolding heat effects of which were systematically higher than those of the WT protein (about 16.4 kJ/mol at 323 K), which may be close to the upper limit of the enthalpy gain due to 10 residue beta-hairpin folding. At the same time, the chimeras with high nose stability, which, like SHH, have a hydrophobic (IVY) cluster on their surface, showed a lower apparent unfolding heat effect, much closer to that of the WT protein. The possible reasons for this difference are discussed.

Crystallographic structure of the SH3 domain of the human c-Yes tyrosine kinase: loop flexibility and amyloid aggregation.

SH3 domains from the Src family of tyrosine kinases represent an interesting example of the delicate balance between promiscuity and specificity characteristic of proline-rich ligand recognition by SH3 domains. The development of inhibitors of therapeutic potential requires a good understanding of the molecular determinants of binding affinity and specificity and relies on the availability of high quality structural information. Here, we present the first high-resolution crystal structure of the SH3 domain of the c-Yes oncogen. Comparison with other SH3 domains from the Src family revealed significant deviations in the loop regions. In particular, the n-Src loop, highly flexible and partially disordered, is stabilized in an unusual conformation by the establishment of several intramolecular hydrogen bonds. Additionally, we present here the first report of amyloid aggregation by an SH3 domain from the Src family.

Structure of human TSG101 UEV domain.

The UEV domain of the TSG101 protein functions in the vacuolar protein-sorting pathway and in the budding process of HIV-1 and other retroviruses by recognizing ubiquitin in proteins tagged for degradation and short sequences in viral proteins containing an essential and well conserved PTAP motif, respectively. A deep understanding of these interactions is key to the rational design of much-needed novel antivirals. Here, the crystal structure of the TSG101 UEV domain (TSG101-UEV) is presented. TSG101-UEV was crystallized in the presence of PEG 4000 and ammonium sulfate. Under these conditions, crystals were obtained in space group R3, with unit-cell parameters a = b = 97.9, c = 110.6 A, alpha = beta = 90, gamma = 120 degrees . Phases were solved by molecular replacement and the crystal structure of TSG101-UEV was refined to an R factor of 18.8% at 2.2 A resolution. A comparison between the crystal structure and previously reported NMR structures has revealed significant differences in the conformation of one of the loops implicated in ubiquitin recognition. Also, the resulting structure has provided information about the presence of water molecules at the binding interface that could be of relevance for peptide recognition.

Role of the surface charges D72 and K8 in the function and structural stability of the cytochrome c from Nostoc sp. PCC 7119.

We investigated the role of electrostatic charges at positions D72 and K8 in the function and structural stability of cytochrome c6 from Nostoc sp. PCC 7119 (cyt c6). A series of mutant forms was generated to span the possible combinations of charge neutralization (by mutation to alanine) and charge inversion (by mutation to lysine and aspartate, respectively) in these positions. All forms of cyt c6 were functionally characterized by laser flash absorption spectroscopy, and their stability was probed by urea-induced folding equilibrium relaxation experiments and differential scanning calorimetry. Neutralization or inversion of the positive charge at position K8 reduced the efficiency of electron transfer to photosystem I. This effect could not be reversed by compensating for the change in global charge that had been introduced by the mutation, indicating a specific role for K8 in the formation of the electron transfer complex between cyt c6 and photosystem I. Replacement of D72 by asparagine or lysine increased the efficiency of electron transfer to photosystem I, but destabilized the protein. D72 apparently participates in electrostatic interactions that stabilize the structure of cyt c6. The destabilizing effect was reduced when aspartate was replaced by the small amino acid alanine. Complementing the mutation D72A with a charge neutralization or inversion at position K8 led to mutant forms of cyt c6 that were more stable than the wild-type under all tested conditions.

Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant.

We have studied the stability of the histone-like, DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant by differential scanning microcalorimetry and CD under acidic conditions at various concentrations within the range of 2-225 micro m of monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation/unfolding model from the folded, dimeric state to the unfolded, monomeric one. The unfolding enthalpy is very low even when taking into account that the two disordered DNA-binding arms probably do not contribute to the cooperative unfolding, whereas the quite small value for the unfolding heat capacity change (3.7 kJ.K(-1).mol(-1)) stabilizes the protein within a broad temperature range, as shown by the stability curves (Gibbs energy functions vs. temperature), even though the Gibbs energy of unfolding is not very high either. The protein is stable at pH 4.00 and 3.75, but becomes considerably less so at pH 3.50 and below, to the point that a simple decrease in concentration will lead to unfolding of both the wild-type and the mutant protein at pH 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJ.mol(-1) at 25 degrees C for DeltaDeltaG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13.

Thermodynamic dissection of the binding energetics of proline-rich peptides to the Abl-SH3 domain: implications for rational ligand design.

The inhibition of the interactions between SH3 domains and their targets is emerging as a promising therapeutic strategy. To date, rational design of potent ligands for these domains has been hindered by the lack of understanding of the origins of the binding energy. We present here a complete thermodynamic analysis of the binding energetics of the p41 proline-rich decapeptide (APSYSPPPPP) to the SH3 domain of the c-Abl oncogene. Isothermal titration calorimetry experiments have revealed a thermodynamic signature for this interaction (very favourable enthalpic contributions opposed by an unfavourable binding entropy) inconsistent with the highly hydrophobic nature of the p41 ligand and the Abl-SH3 binding site. Our structural and thermodynamic analyses have led us to the conclusion, having once ruled out any possible ionization events or conformational changes coupled to the association, that the establishment of a complex hydrogen-bond network mediated by water molecules buried at the binding interface is responsible for the observed thermodynamic behaviour. The origin of the binding energetics for proline-rich ligands to the Abl-SH3 domain is further investigated by a comparative calorimetric analysis of a set of p41-related ligands. The striking effects upon the enthalpic and entropic contributions provoked by conservative substitutions at solvent-exposed positions in the ligand confirm the complexity of the interaction. The implications of these results for rational ligand design are discussed.

A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core.

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.

Characterization of single-tryptophan mutants of histidine-containing phosphocarrier protein: evidence for local rearrangements during folding from high concentrations of denaturant.

We have used site-directed mutagenesis in combination with a battery of biophysical techniques to probe the stability and folding behavior of a small globular protein, the histidine-containing phosphocarrier protein (HPr). Specifically, the four phenylalanine residues (2, 22, 29, and 48) of the wild-type protein were individually replaced by single tryptophans, thus introducing site-specific probes for monitoring the behavior of the protein. The folding of the tryptophan mutants was investigated by NMR, DSC, CD, intrinsic fluorescence, fluorescence anisotropy, and fluorescence quenching. The heat-induced denaturation of all four mutants, and the GdnHCl-induced unfolding curves of F2W, F29W, and F48W, can be fitted adequately to a two-state model, in agreement with the observations for the wild-type protein. The GdnHCl unfolding transitions of F22W, however, showed the accumulation of an intermediate state at low concentrations of denaturant. Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al. (1998) Biochemistry 37, 622-637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS. These observations indicate that the second slow phase reflects a local, rather than a global, rearrangement from a well-structured highly nativelike intermediate state to the fully folded native state that has less hydrophobic surface exposed to the solvent. The detection of the second slow phase by the use of selective labeling of different regions of the protein with fluorophores illustrates the need for an integrated approach in order to understand the intricate details of the folding reactions of even the simplest proteins.

Unfolding and aggregation during the thermal denaturation of streptokinase.

The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.0 and a sample concentration of less than approximately 1.5 mg x mL(-1), or pH 8.0, the heat capacity curves of intact SK can be quantitatively described by three independent two-state transitions, each of which compares well with the two-state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH 7.0 and high sample concentration, or at pH 6.0 at any concentration investigated, the thermal unfolding of domain A was accompanied by the time-dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves, which become concentration dependent and kinetically controlled, and thus precludes their proper analysis by standard deconvolution methods. A simple model involving time-dependent, high-order aggregation may account for the observed effects. Limited-proteolysis experiments suggest that in the aggregates the N-terminal segment 1-63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured. Unfolding of domain A, under conditions where the N-terminal segment 1-63 has a high propensity for beta sheet structure and a partially formed hydrophobic core, gives rise to rapid aggregation. It is likely that this region is able to act as a nucleus for the aggregation of the full-length protein.

The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride.

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been studied. The native structure of the 70-amino-acid-long protein turned out to be extremely stable against heat and denaturant-induced unfolding. At pH 2.5 and low ionic strength, it denatures at 102 degrees C, while at 25 degrees C, the structure only unfolds in 6.3 M guanidinium hydrochloride (GuHCl) and does not unfold even in 8 M urea. A comparison of its thermal unfolding in water and in the presence of urea shows a good correspondence between the two deltaGw(298) values, which are about 30 kJ mol(-1) at pH 2.5 and low ionic strength. The stability of the structure is highly dependent upon ionic strength and so GuHCl acts both as a denaturant and a stabilising agent. This seems to be why the deltaGw(298) value calculated from the unfolding data in GuHCl is twice as high as in the absence of this salt. At least part of the high stability of native AS-48 can almost certainly be put down to its circular organization since other structural features are quite normal for a protein of this size.