SUMMARY The entry of human immunodeficiency virus (HIV-1) into cells is initiated by binding of the gp120 exterior envelope glycoprotein to the receptor, CD4. How does CD4 binding trigger conformational changes in gp120 that allow the gp41 transmembrane envelope glycoprotein to mediate viral-cell membrane fusion? The transition from the unliganded to the CD4-bound state is regulated by two potentially flexible topological layers (“Layers 1 and 2”) in the gp120 inner domain. Both layers apparently contribute to the non-covalent association of unliganded gp120 with gp41. After CD4 makes initial contact with the gp120 outer domain, Layer 1-Layer 2 interactions strengthen gp120-CD4 binding by reducing the off-rate. Layer 1-Layer 2 interactions also destabilize the activated state induced on HIV-1 by treatment with soluble CD4. Thus, despite lack of contact with CD4, the gp120 inner domain layers govern CD4 triggering by participating in conformational transitions within gp120 and regulating the interaction with gp41.

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. ; however, the marked decrease in k cat suggests a role for Arg 101 in catalysis. The V max of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK aes is similar for the R101Q and R120Q enzymes. In contrast, the pK aes for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg 110 and Arg 133 normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.The mitochondrial NADP-specific pig heart isocitrate dehydrogenase (EC 1.1.1.42) catalyzes the divalent metal ion-dependent oxidative decarboxylation of isocitrate to ␣-ketoglutarate, and it is considered that the metal-tribasic isocitrate complex is the preferred substrate (1). The enzyme is a homodimer (2, 3), with a subunit mass of 46,600 Da consisting of 413 amino acids of determined sequence (4). A 13 C-NMR study using specifically enriched isocitrate demonstrated that all three carboxyls of the substrate remain fully ionized from pH 5.5 to 7.5 when bound to the enzyme, although the carboxylates of free isocitrate become protonated over this pH range (5). This result could be due to the presence of positively charged groups in the region of the substrate binding site. The first evidence of the importance of arginines in the function of NADP-dependent isocitrate dehydrogenase came from the inactivation of the pig heart enzyme by 2,3-butanedione (6). A maximum of four arginines were implicated in catalytic activity and, because isocitrate markedly decreased the inactivation rate, it was suggested that at least some of these residues were at or near the isocitrate binding site.

Nonnative disulfide bond formation can play a critical role in the assembly of disulfide bonded proteins. During the folding and assembly of the P22 tailspike protein, nonnative disulfide bonds form both in vivo and in vitro. However, the mechanism and identity of cysteine disulfide pairs remains elusive, particularly for P22 tailspike, which contains no disulfide bonds in its native, functional form. Understanding the interactions between cysteine residues is important for developing a mechanistic model for the role of nonnative cysteines in P22 tailspike assembly. Prior in vivo studies have suggested that cysteines 496, 613, and 635 are the most likely site for sulfhydryl reactivity. Here we demonstrate that these three cysteines are critical for efficient assembly of tailspike trimers, and that interactions between cysteine pairs lead to productive assembly of native tailspike.

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