Crystal structure and ligand binding properties of the D1D2 region of the inhibitory receptor LIR-1 (ILT2).

LIR-1 is an inhibitory receptor that recognizes class I MHC molecules and the human cytomegalovirus class I homolog UL18. Here, we report the 2.1 A resolution crystal structure of the ligand binding portion of LIR-1 (domains 1 and 2 [D1D2]) and localize the binding region for UL18. LIR-1 D1D2 is composed of two immunoglobulin-like domains arranged at an acute angle to form a bent structure resembling the structures of natural killer inhibitory receptors (KIRs). The LIR-1 binding site comprises a portion of D1 distant from the interdomain hinge region that constitutes the KIR binding site, consistent with differences in LIR-1 and KIR recognition properties and functions.

The inhibitory receptor LIR-1 uses a common binding interaction to recognize class I MHC molecules and the viral homolog UL18.

LIR-1 is a class I MHC receptor related to natural killer inhibitory receptors (KIRs). Binding of LIR-1 or KIRs to class I molecules results in inhibitory signals. Unlike individual KIRs, LIR-1 recognizes many class I alleles and also binds UL18, a human cytomegalovirus class I MHC homolog. Here, we show that LIR-1 interacts with the relatively nonpolymorphic alpha3 domain of class I proteins and the analogous region of UL18 using its N-terminal immunoglobulin-like domain. The >1000-fold higher affinity of LIR-1 for UL18 than for class I illustrates how a viral protein competes with host proteins to subvert the host immune response. LIR-1 recognition of class I molecules resembles the CD4-class II MHC interaction more than the KIR-class I interaction, implying a functional distinction between LIR-1 and KIRs.

Characterization of the interaction between the herpes simplex virus type I Fc receptor and immunoglobulin G.

Herpes simplex virus type I (HSV-1) virions and HSV-1-infected cells bind to human immunoglobulin G (hIgG) via its Fc region. A complex of two surface glycoproteins encoded by HSV-1, gE and gI, is responsible for Fc binding. We have co-expressed soluble truncated forms of gE and gI in Chinese hamster ovary cells. Soluble gE-gI complexes can be purified from transfected cell supernatants using a purification scheme that is based upon the Fc receptor function of gE-gI. Using gel filtration and analytical ultracentrifugation, we determined that soluble gE-gI is a heterodimer composed of one molecule of gE and one molecule of gI and that gE-gI heterodimers bind hIgG with a 1:1 stoichiometry. Biosensor-based studies of the binding of wild type or mutant IgG proteins to soluble gE-gI indicate that histidine 435 at the CH2-CH3 domain interface of IgG is a critical residue for IgG binding to gE-gI. We observe many similarities between the characteristics of IgG binding by gE-gI and by rheumatoid factors and bacterial Fc receptors such as Staphylococcus aureus protein A. These observations support a model for the origin of some rheumatoid factors, in which they represent anti-idiotypic antibodies directed against antibodies to bacterial and viral Fc receptors.

Modulation of natural killer cell cytotoxicity in human cytomegalovirus infection: the role of endogenous class I major histocompatibility complex and a viral class I homolog.

Natural killer (NK) cells have been implicated in early immune responses against certain viruses, including cytomegalovirus (CMV). CMV causes downregulation of class I major histocompatibility complex (MHC) expression in infected cells; however, it has been proposed that a class I MHC homolog encoded by CMV, UL18, may act as a surrogate ligand to prevent NK cell lysis of CMV-infected cells. In this study, we examined the role of UL18 in NK cell recognition and lysis using fibroblasts infected with either wild-type or UL18 knockout CMV virus, and by using cell lines transfected with the UL18 gene. In both systems, the expression of UL18 resulted in the enhanced killing of target cells. We also show that the enhanced killing is due to both UL18-dependent and -independent mechanisms, and that the killer cell inhibitory receptors (KIRs) and CD94/NKG2A inhibitory receptors for MHC class I do not play a role in affecting susceptibility of CMV-infected fibroblasts to NK cell-mediated cytotoxicity.

Characterization of a murine cytomegalovirus class I major histocompatibility complex (MHC) homolog: comparison to MHC molecules and to the human cytomegalovirus MHC homolog.

Both human and murine cytomegaloviruses (HCMV and MCMV) down-regulate expression of conventional class I major histocompatibility complex (MHC) molecules at the surfaces of infected cells. This allows the infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to natural killer cells, which lyse cells that lack class I molecules. Both HCMV and MCMV encode class I MHC heavy-chain homologs that may function in immune response evasion. We previously showed that a soluble form of the HCMV class I homolog (U(L)18) expressed in Chinese hamster ovary cells binds the class I MHC light-chain beta2-microglobulin and a mixture of endogenous peptides (M. L. Fahnestock, J. L. Johnson, R. M. R. Feldman, J. M. Neveu, W. S. Lane, and P. J. Bjorkman, Immunity 3:583-590, 1995). Consistent with this observation, sequence comparisons suggest that U(L)18 contains the well-characterized groove that serves as the binding site in MHC molecules for peptides derived from endogenous and foreign proteins. By contrast, the MCMV homolog (m144) contains a substantial deletion within the counterpart of its alpha2 domain and might not be expected to contain a groove capable of binding peptides. We have now expressed a soluble version of m144 and verified that it forms a heavy chain-beta2-microglobulin complex. By contrast to U(L)18 and classical class I MHC molecules, m144 does not associate with endogenous peptides yet is thermally stable. These results suggest that U(L)18 and m144 differ structurally and might therefore serve different functions for their respective viruses.

The role of Pro20 in the isomerization of myotoxin a from Crotalus viridis viridis: folding and structural characterization of synthetic myotoxin a and its Pro20Gly homolog.

Biochemical characterization has established the presence of two conformational forms of myotoxin a. To test the hypothesis that this may be due to cis-trans isomerization at Pro20, synthetic versions of myotoxin a and its Pro20-->Gly structural homolog were folded, then purified using a two-step cation-exchange/reverse-phase perfusion chromatography method. The disulfide bond configuration for the folded proteins was found to be the same as that of native myotoxin a. CE and RPHPLC revealed that folded synthetic myotoxin a exists in two conformations while the Pro20-->Gly homolog exists in only one, supporting the hypothesis that cis-trans isomerization at Pro20 is the source of the myotoxin a conformational heterogeneity.

Stereochemical probes of the argininosuccinate synthetase reaction.

The stereochemical course of the argininosuccinate synthetase reaction has been determined. The SP isomer of [alpha-17O,alpha-18O,alpha beta-18O]ATP is cleaved to (SP)-[16O,17O,18O]AMP by the action of argininosuccinate synthetase in the presence of citrulline and aspartate. The overall stereochemical transformation is therefore net inversion, and thus the enzyme does not catalyze the formation of an adenylylated enzyme intermediate prior to the synthesis of citrulline adenylate. The RP isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) is a substrate in the presence of Mg2+, but the SP isomer is a substrate when Cd2+ is used as the activating divalent cation. Therefore, the lambda screw sense configuration of the beta,gamma-bidentate metal--ATP complex is preferred by the enzyme as the actual substrate. No significant discrimination could be detected between the RP and SP isomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) when Mg2+ or Mn2+ are used as the divalent cation. Argininosuccinate synthetase has been shown to require a free divalent cation for full activity in addition to the metal ion needed to complex the ATP used in the reaction.