The three-dimensional structure of the ligand-binding region of human E-selectin has been determined at 2.0 A resolution. The structure reveals limited contact between the two domains and a coordination of Ca2+ not predicted from other C-type lectins. Structure/function analysis indicates a defined region and specific amino-acid side chains that may be involved in ligand binding. These features of the E-selectin/ligand interaction have important implications for understanding the recruitment of leukocytes to sites of inflammation.

The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [alpha-2-O-methyl-, 4-O-acetyl-alpha-2-O-methyl-, 9-amino-9-deoxy-alpha-2-O-methyl-, and alpha-2-O-(4'-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (alpha 2-3)sialyllactose, and suggest the location of an alpha-glycosidic chain (4'-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with alpha-2-O-methylsialic acid contains the mutation Gly-135-->Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.

Although the murine retrovirus SL3-3 is highly leukaemogenic, in both the structure of its genome and in its properties of replication in tissue culture it closely resembles the nonleukaemogenic retrovirus Akv (refs 3, 4). An earlier investigation of the properties of recombinant SL3-3-Akv viruses localized the major determinant of leukaemogenicity outside the env gene, in a region of the viral genome that includes the gag gene and the noncoding long terminal repeat (LTR). To localize the determinant of SL3-3's leukaemogenicity more precisely we have now construced a recombinant provirus containing the LTR of SL3-3 and the coding region of Akv. The leukaemogenicity of these recombinants demonstrates that the determinant of leukaemogenicity lies within the SL3-3 LTR. Nucleotide sequencing of the LTRs of SL3-3 and Akv shows that they differ by a set of changes in the region thought to contain a transcriptional enhancer element. We suggest that enhancer region sequences are the major determinants of leukaemogenicity in these viruses.

The sequence of 2,191 nucleotides encoding the env gene of murine retrovirus Akv was determined by using a molecular clone of the Akv provirus. Deduction of the encoded amino acid sequence showed that a single open reading frame encodes a 638-amino acid precursor to gp70 and p15E. In addition, there is a typical leader sequence preceding the amino terminus of gp70. The locations of potential glycosylation sites and other structural features indicate that the entire gp70 molecule and most of p15E are located on the outer side of the membrane. Internal cleavage of the env precursor to generate gp70 and p15E occurs immediately adjacent to several basic amino acids at the carboxyl terminus of gp70. This cleavage generates a region of 42 uncharged, relatively hydrophobic amino acids at the amino terminus of p15E, which is located in a position analogous to the hydrophobic membrane fusion sequence of influenza virus hemagglutinin. The mature polypeptides are predicted to associate with the membrane via a region of 30 uncharged, mostly hydrophobic amino acids located near the carboxyl terminus of p15E. Distal to this membrane association region is a sequence of 35 amino acids at the carboxyl terminus of the env precursor, which is predicted to be located on the inner side of the membrane. By analogy to Moloney murine leukemia virus, a proteolytic cleavage in this region removes the terminal 19 amino acids, thus generating the carboxyl terminus of p15E. This leaves 15 amino acids at the carboxyl terminus of p15E on the inner side of the membrane in a position to interact with virion cores during budding. The precise location and order of the large RNase T 1 -resistant oligonucleotides in the env region were determined and compared with those from several leukemogenic viruses of AKR origin. This permitted a determination of how the differences in the leukemogenic viruses affect the primary structure of the env gene products.

No abstract

No abstract

Molecular features of ligand binding to MHC class II HLA-DR molecules have been elucidated through a combination of peptide structure-activity studies and structure-based drug design, resulting in analogues with nanomolar affinity in binding assays. Stabilization of lead compounds against cathepsin B cleavage by N-methylation of noncritical backbone NH groups or by dipeptide mimetic substitutions has generated analogues that compete effectively against protein antigens in cellular assays, resulting in inhibition of T-cell proliferation. Crystal structures of four ternary complexes of different peptide mimetics with the rheumatoid arthritis-linked MHC DRB10401 and the bacterial superantigen SEB have been obtained. Peptide-sugar hybrids have also been identified using a structure-based design approach in which the sugar residue replaces a dipeptide. These studies illustrate the complementary roles played by phage display library methods, peptide analogue SAR, peptide mimetics substitutions, and structure-based drug design in the discovery of inhibitors of antigen presentation by MHC class II HLA-DR molecules.

We have chosen to discuss the clinical and etiologic heterogeneity of diabetes in man, since in recent years there has been an accumulation of evidence from many centers, including our own, that idiopathic or primary diabetes mellitus is not a single disease entity. What we call diabetes mellitus appears to consist of a number of heterogeneous syndromes characterized by a continuum of metabolic changes secondary to insufficient insulin action and by various tissue changes referred to as the chronic complications of diabetes.