The solution structure of the ectodomain of simian immunodeficiency virus (SIV) gp41 (e-gp41), consisting of residues 27-149, has been determined by multidimensional heteronuclear NMR spectroscopy. SIV e-gp41 is a symmetric 44 kDa trimer with each subunit consisting of antiparallel N-terminal (residues 30-80) and C-terminal (residues 107-147) helices connected by a 26 residue loop (residues 81-106). The N-terminal helices of each subunit form a parallel coiled-coil structure in the interior of the complex which is surrounded by the C-terminal helices located on the exterior of the complex. The loop region is ordered and displays numerous intermolecular and non-sequential intramolecular contacts. The helical core of SIV e-gp41 is similar to recent X-ray structures of truncated constructs of the helical core of HIV-1 e-gp41. The present structure establishes unambiguously the connectivity of the N-and C-terminal helices in the trimer, and characterizes the conformation of the intervening loop, which has been implicated by mutagenesis and antibody epitope mapping to play a key role in gp120 association. In conjunction with previous studies, the solution structure of the SIV e-gp41 ectodomain provides insight into the binding site of gp120 and the mechanism of cell fusion. The present structure of SIV e-gp41 represents one of the largest protein structures determined by NMR to date.

The solution structure of the N-terminal zinc binding domain (residues 1-55; IN1-55) of HIV-1 integrase has been solved by NMR spectroscopy. IN1-55 is dimeric, and each monomer comprises four helices with the zinc tetrahedrally coordinated to His 12, His 16, Cys 40 and Cys 43. IN1-55 exists in two interconverting conformational states that differ with regard to the coordination of the two histidine side chains to zinc. The different histidine arrangements are associated with large conformational differences in the polypeptide backbone (residues 9-18) around the coordinating histidines. The dimer interface is predominantly hydrophobic and is formed by the packing of the N-terminal end of helix 1, and helices 3 and 4. The monomer fold is remarkably similar to that of a number of helical DNA binding proteins containing a helix-turn-helix (HTH) motif with helices 2 and 3 of IN1-55 corresponding to the HTH motif. In contrast to the DNA binding proteins where the second helix of the HTH motif is employed for DNA recognition, IN1-55 uses this helix for dimerization.

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We have determined the solution NMR structure of a recombinant peptide that consists of the first 156 residues of erythroid ␣-spectrin. The first 20 residues preceding the first helix (helix C) are in a disordered conformation. The subsequent three helices (helices A 1 , B 1 , and C 1 ) form a triple helical bundle structural domain that is similar, but not identical, to previously published structures for spectrin from Drosophila and chicken brain. Paramagnetic spin label-induced NMR resonance broadening shows that helix C, the partial domain involved in ␣-and ␤-spectrin association, exhibits little interaction with the structural domain. Surprisingly, helix C is connected to helix A 1 of the structural domain by a segment of 7 residues (the junction region) that exhibits a flexible disordered conformation, in contrast to the predicted rigid helical structure. We suggest that the flexibility of this particular junction region may play an important role in modulating the association affinity of ␣-and ␤-spectrin at the tetramerization site of different isoforms, such as erythroid spectrin and brain spectrin. These findings may provide insight for explaining various physiological and pathological conditions that are a consequence of varying ␣-and ␤-subunit self-association affinities in their formation of the various spectrin tetramers.Spectrin, a member of the spectrin superfamily and a major protein in the membrane (cyto)skeleton, is ubiquitous among vertebrate tissues, as well as in simple metazoans, implying that spectrin plays a fundamental role in cells (1-3). After first being identified in erythrocytes (4), many distinct spectrin isoforms have since been discovered. In humans, two ␣-spectrin subunits (␣I and ␣II), four ␤-spectrin subunits (␤I, ␤II, ␤III, and ␤IV), and a ␤-H subunit have been sequenced (1). Similar isoforms in mice have also been studied (5). Alternative splicing (e.g. see Ref. 6) adds additional diversity among ␣-and ␤-spectrin isoforms. Many functions of different spectrin isoforms involve interactions with other molecules such as spectrinactin interaction, spectrin-membrane interaction, spectrin-ion channel interaction, etc. (1). Yet some of the most fundamental functions of spectrin involve spectrin "self-association." The activity of spectrin in stabilizing cell-cell contacts and in achieving normal columnar epithelial cell shape requires the formation of tetramers (1,7,8). Spectrin tetramers have been suggested to be cooperatively coupled to membrane assembly (9). Many hereditary hemolytic anemias involve single amino acid mutations in erythrocyte spectrin that destabilize its tetramers, resulting in low levels of spectrin tetramers and high levels of dimers (9 -11). Thus, the tetramerization site is an important functional site for most spectrins.In erythrocyte spectrin (␣I/␤I), ␣-and ␤-spectrin associate with relatively high affinity (nM K d values) at the N-terminal end of the ␤-spectrin and the C-terminal end of the ␣-spectrin (dimer nucleation site) to give ␣␤ heterodimers (12, 1...

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.

In human immunodeficiency virus (HIV) the viral envelope proteins gp41 and gp120 form a non-covalent complex, which is a potential target for AIDS therapies. In addition gp41 plays a possible role in HIV infection of B cells via the complement system. In an effort to better understand the molecular interactions of gp41, the structure of the HIV gp41 ectodomain has been modeled using the NMR restraints of the simian immunodeficiency virus (SIV) gp41 ectodomain (M. Caffrey, M. Cai, J. Kaufman, S.J. Stahl, P.T. Wingfield, A.M. Gronenborn, G.M. Clore, Solution structure of the 44 kDa ectodomain of SIV gp41, EMBO J. 17 (1998) 4572--4584). The resulting model presents the first structural information for the HIV gp41 loop, which has been implicated to play a direct role in binding to gp120 and C1q of the complement system.

Severe acute respiratory syndrome coronavirus (SARS-CoV) and Ebola, Hendra, and Nipah viruses are members of different viral families and are known causative agents of fatal viral diseases. These viruses depend on cathepsin L for entry into their target cells. The viral glycoproteins need to be primed by protease cleavage, rendering them active for fusion with the host cell membrane. In this study, we developed a novel high-throughput screening assay based on peptides, derived from the glycoproteins of the aforementioned viruses, which contain the cathepsin L cleavage site. We screened a library of 5,000 small molecules and discovered a small molecule that can inhibit the cathepsin L cleavage of all viral peptides with minimal inhibition of cleavage of a host protein-derived peptide (pro-neuropeptide Y). The small molecule inhibited the entry of all pseudotyped viruses in vitro and the cleavage of SARS-CoV spike glycoprotein in an in vitro cleavage assay. In addition, the Hendra and Nipah virus fusion glycoproteins were not cleaved in the presence of the small molecule in a cell-based cleavage assay. Furthermore, we demonstrate that the small molecule is a mixed inhibitor of cathepsin L. Our broad-spectrum antiviral small molecule appears to be an ideal candidate for future optimization and development into a potent antiviral against SARS-CoV and Ebola, Hendra, and Nipah viruses. IMPORTANCEWe developed a novel high-throughput screening assay to identify small molecules that can prevent cathepsin L cleavage of viral glycoproteins derived from SARS-CoV and Ebola, Hendra, and Nipah viruses that are required for their entry into the host cell. We identified a novel broad-spectrum small molecule that could block cathepsin L-mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from SARS-CoV or Ebola, Hendra, or Nipah virus. The small molecule can be further optimized and developed into a potent broad-spectrum antiviral drug.

Background: ZO-1 is a scaffolding protein implicated in the assembly of tight junctions. Results: Structures of core PDZ-SH3-GUK, plus and minus JAM-A peptide, and isolated PDZ are presented. Conclusion: The SH3 domain is required for JAM-A binding to PDZ3. Significance: This is the first demonstration for the role of an adjacent domain to the binding of ligands to PDZ domains in the MAGUK family.