Intrahost evolution of the HIV-2 capsid correlates with progression to AIDS.

HIV-2 infection will progress to AIDS in most patients without treatment, albeit at approximately half the rate of HIV-1 infection. HIV-2 capsid (p26) amino acid polymorphisms are associated with lower viral loads and enhanced processing of T cell epitopes, which may lead to protective Gag-specific T cell responses common in slower progressors. Lower virus evolutionary rates, and positive selection on conserved residues in HIV-2 env have been associated with slower progression to AIDS. In this study we analysed 369 heterochronous HIV-2 p26 sequences from 12 participants with a median age of 30 years at enrolment. CD4% change over time was used to stratify participants into relative faster and slower progressor groups. We analysed p26 sequence diversity evolution, measured site-specific selection pressures and evolutionary rates, and determined if these evolutionary parameters were associated with progression status. Faster progressors had lower CD4% and faster CD4% decline rates. Median pairwise sequence diversity was higher in faster progressors (5.7x10-3 versus 1.4x10-3 base substitutions per site, P<0.001). p26 evolved under negative selection in both groups (dN/dS=0.12). Median virus evolutionary rates were higher in faster than slower progressors - synonymous rates: 4.6x10-3 vs. 2.3x10-3; and nonsynonymous rates: 6.9x10-4 vs. 2.7x10-4 substitutions/site/year, respectively. Virus evolutionary rates correlated negatively with CD4% change rates (ρ = -0.8, P=0.02), but not CD4% level. The signature amino acid at p26 positions 6, 12 and 119 differed between faster (6A, 12I, 119A) and slower (6G, 12V, 119P) progressors. These amino acid positions clustered near to the TRIM5α/p26 hexamer interface surface. p26 evolutionary rates were associated with progression to AIDS and were mostly driven by synonymous substitutions. Nonsynonymous evolutionary rates were an order of magnitude lower than synonymous rates, with limited amino acid sequence evolution over time within hosts. These results indicate HIV-2 p26 may be an attractive therapeutic target.

Association of LILRA2 (ILT1, LIR7) splice site polymorphism with systemic lupus erythematosus and microscopic polyangiitis.

Leukocyte immunoglobulin-like receptors (LILRs) are inhibitory, stimulatory or soluble receptors encoded within the leukocyte receptor complex. Some LILRs are extensively polymorphic, and exhibit evidence for balancing selection and association with disease susceptibility. LILRA2 (LIR7/ILT1) is an activating receptor highly expressed in inflammatory tissues, and is involved in granulocyte and macrophage activation. In this study, we examined the association of LILRA2 and adjacently located LILRA1 with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and microscopic polyangiitis (MPA). Polymorphism screening detected a LILRA2 SNP (rs2241524 G>A) that disrupts splice acceptor site of intron 6. Case-control association studies on 273 Japanese SLE, 296 RA, 50 MPA and 284 healthy individuals revealed increase of genotype A/A in SLE (12.1%, odds ratio (OR) 1.82, 95% confidence interval (CI) 1.02-3.24, P=0.041) and in MPA (16.0%, OR 2.52, 95% CI 1.07-5.96, P=0.049) compared with healthy individuals (7.0%). The risk allele caused an activation of a cryptic splice acceptor site that would lead to a novel LILRA2 isoform lacking three amino acids in the linker region (Delta 419-421). Flow cytometry indicated that this isoform was expressed on the surface of monocytes. These findings suggested that LILRA2 Delta 419-421 isoform encoded by the splice site SNP may play a role in SLE and MPA.

The human low affinity Fcgamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties.

Fcgamma receptors (FcgammaRs) are expressed on all immunologically active cells. They bind the Fc portion of IgG, thereby triggering a range of immunological functions. We have used surface plasmon resonance to analyze the kinetic and thermodynamic properties of the interactions between the ectodomains of human low affinity FcgammaRs (FcgammaRIIa, FcgammaRIIb, and FcgammaRIIIb-NA2) and IgG1 or the Fc fragment of IgG1. All three receptors bind Fc or IgG with similarly low affinities (K(D) approximately 0.6-2.5 microm) and fast kinetics, suggesting that FcgammaR-mediated recognition of aggregated IgG and IgG-coated particles or cells is mechanistically similar to cell-cell recognition. Interestingly, the Fc receptors exhibit distinct thermodynamic properties. Whereas the binding of the FcgammaRIIa and FcgammaRIIb to Fc is driven by favorable entropic and enthalpic changes, the binding of FcgammaRIII is characterized by highly unfavorable entropic changes. Although the structural bases for these differences remain to be determined, they suggest that the molecular events coupled to the binding differ among the low affinity FcgammaRs.

Detection of autoantibodies to killer immunoglobulin-like receptors using recombinant fusion proteins for two killer immunoglobulin-like receptors in patients with systemic autoimmune diseases.

OBJECTIVE: To investigate the existence of autoantibodies to killer immunoglobulin-like receptors (KIRs), especially p58.1 (KIR2DL1) and p58.2 (KIR2DL3), in patients with systemic autoimmune diseases. METHODS: Sera from 30 patients with systemic lupus erythematosus (SLE), 30 patients with rheumatoid arthritis (RA), 22 patients with Behçet's disease, and 20 healthy control subjects were tested for anti-p58.1 and anti-p58.2 antibodies by Western blot analysis using recombinant p58.1 and p58.2 proteins. Furthermore, clinical features and laboratory data were compared between the anti-p58.1/58.2 antibody-positive and -negative patients. RESULTS: Anti-p58.1 antibodies were detected in 7 (23.3%) of the 30 patients with SLE, 9 (30%) of the 30 patients with RA, and 6 (27.3%) of the 22 patients with Behçet's disease. Anti-p58.2 antibodies were detected in the same 22 patients who were positive for the anti-p58.1 antibodies. None of the serum samples from the healthy donors were positive for antibodies to the recombinant p58.1 or p58.2 molecules. Compared with the anti-p58.1/ 58.2 antibody-negative patients, the anti-p58.1/58.2 antibody-positive patients had significantly elevated levels of serum IgG in all 3 diseases tested, an accelerated erythrocyte sedimentation rate in RA and SLE, and decreased white blood cell counts in RA. CONCLUSION: This report is the first to describe the presence of autoantibodies to KIR2DL (p58.1 and p58.2) in the sera of patients with systemic autoimmune diseases. Considering the correlation with several clinical features, these autoantibodies may be involved in the pathologic process of the autoimmune diseases.

Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes.

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.

Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha.

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.

MHC superfamily structure and the immune system.

During the past year, a plethora of structural information has provided detailed insights into the interactions between classical MHC class I molecules and their cognate receptors on T cells. Likewise, there have been major advances in our knowledge of the structures and functions of five nonclassical MHC-like molecules: HLA-DM (murine H2-M), HLA-E, HFE, ZAG and MIC-A.

Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties.

Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.

Crystal structure of the human p58 killer cell inhibitory receptor (KIR2DL3) specific for HLA-Cw3-related MHC class I.

BACKGROUND: T cells and natural killer (NK) cells perform complementary roles in the cellular immune system. T cells identify infected cells directly through recognition of antigenic peptides that are displayed at the target cell surface by the classical major histocompatibility complex (MHC) class I molecules. NK cells monitor the target cell surface for malfunction of this display system, lysing potentially infected cells that might otherwise evade recognition by the T cells. Human killer cell inhibitory receptors (KIRs) control this process by either inhibiting or activating the cytotoxic activity of NK cells via specific binding to MHC class I molecules on the target cell. RESULTS: We report the crystal structure of the extracellular region of the human p58 KIR (KIR2DL3), which is specific for the human MHC class I molecule HLA-Cw3 and related alleles. The structure shows the predicted topology of two tandem immunoglobulin-like domains, but comparison with the previously reported structure of the related receptor KIR2DL1 reveals an unexpected change of 23 degrees in the relative orientation of these domains. CONCLUSIONS: The altered orientation of the immunoglobulin-like domains maintains an unusually acute interdomain elbow angle, which therefore appears to be a distinctive feature of the KIRs. The putative MHC class I binding site is located on the outer surface of the elbow, spanning both domains. The unexpected observation that this binding site can be modulated by differences in the relative domain orientations has implications for the general mechanism of KIR-MHC class I complex formation.

Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2).

Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average Mr = 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6122 or P6522) with lattice constants a = b = 95.3, c = 130.8 A. A native data set (3 A resolution) has been collected at the Photon Factory (lambda = 1.0 A).

Structural and functional effect of Trp-62-->Gly and Asp-101-->Gly substitutions on substrate-binding modes of mutant hen egg-white lysozymes.

In order to clarify the structural role of subsite B of hen egg-white lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62-->Gly and Asp-101-->Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-d-glucosamine)3 [(GlcNAc)3], using X-ray crystallography. The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc)3 molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)2, the reducing end of which is an alpha-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a beta-anomer-retaining manner, and so the alpha-anomer product was converted from the beta-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.

Structural analysis of mutant hen egg-white lysozyme preferring a minor binding mode.

Trp62 in hen egg-white lysozyme has general features observed in protein-carbohydrate interactions, a stacking interaction toward nonpolar surface of the substrate sugar residue B and a hydrogen bonding network with the residue C. Our previous report (I. Kumagai, K. Maenaka, F. Sunada, S. Takeda, K. Miura, Eur. J. Biochem. 212 (1993) 151-156.) showed that the substitution of Trp62 changed the substrate binding modes; especially, the Trp62His mutant exhibited the drastic change of the binding mode and preferred to a minor binding mode of the wild-type enzyme. In order to clarify the relationship between functional and structural changes of the Trp62His mutant, we analyzed the structure of the Trp62His mutant hen lysozyme complexed with the substrate analogue, (GlcNAc)3, by X-ray crystallography. The overall protein structure in the mutant lysozyme complex was almost identical to that in the wild-type. His62 shared almost the same plane as the indole ring of Trp62 of the wild-type. Although the (GlcNAc)3 molecule which is an inhibitor against the wild-type lysozyme was cocrystallized, the Trp62His mutant did not put it in the sites A-B-C but hydrolyzed it as a substrate. One of the products, (GlcNAc)2, whose reducing end is alpha-anomer, was bound in another binding mode sticking out from the active-site cleft. The hydrolytic activity against the synthetic substrate showed that the mutant was a beta-anomer retaining enzyme, so the alpha-anomer product was converted from the beta-anomer product. Therefore, the Trp62His mutant showed the remarkable change of the substrate binding modes not by alteration of the catalytic system but possibly by subtle rearrangement of general features of protein-carbohydrate interactions between His62 and the sugar residues B and C.

The 1.8-A X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding.

The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E. coli PotD protein complexed with spermidine was solved at 1.8 A resolution and revealed the detailed substrate-binding mechanism. The structure provided the detailed conformation of the bound spermidine. Furthermore, a water molecule was clearly identified in the binding site lying between the amino-terminal domain and carboxyl-terminal domain. Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine. To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure. The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine. On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.

A stable phage-display system using a phagemid vector: phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline, phosphatase, and anti-HEL monoclonal antibody, HyHEL10.

A stable expression system for displaying the pIII fusion protein on the surface of a filamentous phage was constructed. A phagemid pIII display vector, pLUCK, was constructed by inserting the gene encoding the pIII fusion protein in the opposite direction to that of the lac promoter of pTZ18U. Using this phage display system, two enzymes, hen egg-white lysozyme (HEL) and E. coli alkaline phosphatase, and the single-chain Fv fragment of anti-HEL monoclonal antibody HyHEL10, could be stably and functionally displayed. Northern and primer extension analyses showed that a small amount of the sense mRNA encoding pIII-fused HEL was transcribed from the minor phage promoter located in the region encoding the C-terminus of pIII. Repressed expression of the pIII fusion protein can lead to the display of a wide range of proteins on filamentous phages without the need for strict expression conditions.

Dissection of protein-carbohydrate interactions in mutant hen egg-white lysozyme complexes and their hydrolytic activity.

Trp62 in the binding subsite B of hen egg-white lysozyme shows general features often observed in protein-carbohydrate interactions including a stacking interaction and a hydrogen bonding network with water molecules. A previous report by our group showed that the perturbation of these interactions by substitution of Trp62 with tyrosine or phenylalanine affects the substrate binding modes and also enhances the hydrolytic activity. In order to elucidate the relationship between structural and functional changes of these protein-carbohydrate interactions, the Trp62Tyr and Trp62Phe mutants complexed with the substrate analogue, (GlcNAc)3, were analyzed at 1.8 A resolution by X-ray crystallography. The overall structures of the mutant enzymes are indistinguishable from that of the wild type enzyme. Although the wild-type enzyme binds (GlcNAc)3 in only one binding mode (A-B-C), the Trp62Tyr mutant binds (GlcNAc)3 in two binding modes (A-B-C, B-C-D) and the Trp62Phe mutant has an even weaker binding mode. The aromatic rings of Tyr62 and Phe62 maintain their interactions with the carbohydrate molecules, but make fewer stacking interactions with the GlcNAc in the B site than the wild-type enzyme does. The hydroxyl group of Tyr62 interacts weakly with a water molecule which mediates hydrogen bonding in the GlcNAc residues in the B and C sites. The C-6 hydroxyl group of the GlcNAc residue in the C site rotates around the C-5-C-6 bond to complete the hydrogen bond network in the Trp62Tyr mutant-(GlcNAc)3 complex. On the other hand, this hydrogen bonding network does not form in the Trp62Phe mutant-(GlcNAc)3. In addition to these structural studies, the kinetic parameters of the hydrolysis of 4-methylumbelliferyl N-acetyl-chitotriose, ((GlcNAc)3-MeU), have been determined in order to further characterize the enzymatic properties of these mutant lysozymes. This demonstrates that the modulation of the hydrogen bonding network, including the flexible part of the carbohydrate and water molecules and/or the slight reduction of stacking interaction in the B site, alters the binding mode toward the carbohydrate and induces an enhancement of the hydrolytic activity.

Structural changes of active site cleft and different saccharide binding modes in human lysozyme co-crystallized with hexa-N-acetyl-chitohexaose at pH 4.0.

Human lysozyme was co-crystallized with hexa-N-acetyl-chitohexaose, (GlcNAc)6, at pH 4.0 and 4.0 degrees C in a new orthorhombic form, where two protein molecules, MOL1 and MOL2, were contained in an asymmetric unit. The three-dimensional structure was refined to an R-factor of 17.0% at 1.6 A resolution. It was found that (GlcNAc)6 had already been cleaved to (GlcNAc)4 and (GlcNAc)2. In MOL1, (GlcNAc)4 was bound to the A, B, C, and D subsites, and binding sites of (GlcNAc)2 were close to the E and F subsites proposed on the basis of model building by Phillips and his colleagues. In MOL2, only the (GlcNAc)4 moiety could be found in the A, B, C and D subsites. Significant shifts of the backbone atoms were observed in the region of residues 102 to 120, which composed one side of the wall of the active site cleft. Consequently, the active cleft, with respect to the saccharide binding sites A, B and C, is narrower in both protein molecules. The residues 109 to 111 in site D of MOL1 are moved toward saccharide residue D, whereas those of MOL2 are only slightly shifted. In spite of these facts, the saccharide residues in site MOL1 and MOL2 are moved inside of the cleft. The distribution of water molecules and the hydrogen bond network in site D differ between the structures of MOL1 and MOL2. These structural changes in the active site cleft may be responsible for accommodating the substrate and releasing the products of hydrolysis. These results suggest that the three-dimensional structures of MOL1 and MOL2 remain in intermediate states between a transition state and an enzyme/product complex state.

Contribution to antibody-antigen interaction of structurally perturbed antigenic residues upon antibody binding.

For elucidating the contribution of structurally perturbed antigenic residues upon antibody binding to antigen-antibody interaction, the interaction between hen egg white lysozyme (HEL) and HyHEL10 Fv fragment, which is one of several monoclonal antibodies against HEL and structurally well defined (Padlan, E.A., Silverton, E. W., Sheriff, S., Cohen, G. H., Smith-Gill, S. J., and Davies, D. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5938-5942), was investigated. Asp-101 and Trp-62 of HEL, whose conformations are perturbed by the binding of antibody HyHEL10 in this interaction, were replaced with Gly, and the resulting interactions were studied by assay of the inhibition of the lysozyme activity with the Fv fragment and by titration calorimetry. The results can be summarized as follows. 1) It was possible to prepare the fully functional Fv fragment of HyHEL10 using a secretory expression system in Escherichia coli. Its inhibition profile for HEL activity was almost indistinguishable from that of HyHEL10 IgG, and the contribution of enthalpy to driving the interaction was shown to be significant. 2) A thermodynamic study of the interaction between the D101G mutant HEL and the Fv fragment revealed that, although the negative enthalpy change was smaller than that for the wild type, the Gibbs energy was almost identical to that of the wild type, which resulted from the smaller entropy loss. 3) Study of the interaction between the W62G mutant HEL and this Fv fragment indicated that the rotation of the Trp-62 indole ring upon binding of the antibody made an enthalpic contribution to antibody-antigen interaction, although Trp-62 of HEL was proposed not to be the direct contact residue in the HyHEL10.HEL complex. 4) From these results, it was confirmed experimentally that structural perturbations of antigenic residues upon antibody binding of antigen would contribute to the gain of enthalpic energy, in spite of partial offset by entropic loss, and to driving the interaction.