Stephen C. Harrison

The four-protein Ndc80 complex, an essential kinetochore component conserved from yeast to humans, plays an indispensable role in proper chromosome alignment and segregation during mitosis. In higher eukaryotes, the homologous complex probably resides in the middle domain of the trilaminar kinetochore, linking centromeric heterochromatin with microtubule-associated structures. We have prepared recombinant Ndc80 complex by pairwise coexpression of its components (Ndc80p and Nuf2p; Spc24p and Spc25p) and shown that they form independently stable subcomplexes. Rotary shadowing electron microscopy, combined with limited proteolysis and antibody labeling, demonstrates that the heterotetrameric Ndc80 complex is an ≈570-Å-long rod, with globular regions at either end. The shaft contains α-helical coiled-coil segments from each of the two subcomplexes, linked end-to-end. When integrated with published observations derived from inactivating the components of Ndc80, the molecular organization we deduce suggests that the Spc24p/Spc25p end of the rod faces the centromere and the Ndc80p/Nuf2p end faces a spindle microtubule.

The α/β T cell receptor complex transmits signals from MHC/peptide antigens through a set of constitutively associated signaling molecules, including CD3-ε/γ and CD3-ε/δ. We report the crystal structure at 1.9-Å resolution of a complex between a human CD3-ε/δ ectodomain heterodimer and a single-chain fragment of the UCHT1 antibody. CD3-ε/δ and CD3-ε/γ share a conserved interface between the Ig-fold ectodomains, with parallel packing of the two G strands. CD3-δ has a more electronegative surface and a more compact Ig fold than CD3-γ; thus, the two CD3 heterodimers have distinctly different molecular surfaces. The UCHT1 antibody binds near an acidic region of CD3-ε opposite the dimer interface, occluding this region from direct interaction with the TCR. This immunodominant epitope may be a uniquely accessible surface in the TCR/CD3 complex, because there is overlap between the binding site of the UCHT1 and OKT3 antibodies. Determination of the CD3-ε/δ structure completes the set of TCR/CD3 globular ectodomains and contributes information about exposed CD3 surfaces.

The crystallographically determined structure of the reovirus outer capsid protein σ3 reveals a two‐lobed structure organized around a long central helix. The smaller of the two lobes includes a CCHC zinc‐binding site. Residues that vary between strains and serotypes lie mainly on one surface of the protein; residues on the opposite surface are conserved. From a fit of this model to a reconstruction of the whole virion from electron cryomicroscopy, we propose that each σ3 subunit is positioned with the small lobe anchoring it to the protein μ1 on the surface of the virion, and the large lobe, the site of initial cleavages during entry‐related proteolytic disassembly, protruding outwards. The surface containing variable residues faces solvent. The crystallographic asymmetric unit contains two σ3 subunits, tightly associated as a dimer. One broad surface of the dimer has a positively charged surface patch, which extends across the dyad. In infected cells, σ3 binds dsRNA and inhibits the interferon response. The location and extent of the positively charged surface patch suggest that the dimer is the RNA‐binding form of σ3.

We have used phage-displayed peptide libraries to identify novel ligands to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120. Screening of libraries of random 12-mers, 7-mers, and cyclic 9-mers produced two families of gp120 binding peptides. Members of a family with the prototype sequence RINNIPWSEAMM (peptide 12p1) inhibit the interaction between gp120 and both four-domain soluble CD4 (4dCD4) and monoclonal antibody (MAb) 17b, a neutralizing antibody that covers the chemokine receptor binding surface on gp120. Peptide 12p1 inhibits the interaction of 4dCD4 with gp120 from three different HIV strains, implying that it binds to a conserved site on gp120. Members of a second family of peptides, with the prototype sequence TSPYEDWQTYLM (peptide 12p2), bind more weakly to gp120. They do not detectably affect its interaction with 4dCD4, but they enhance its binding to MAb 17b. A common sequence motif in the two peptide families and cross-competition for gp120 binding suggest that they have overlapping contacts. Their divergent effects on the affinity of gp120 for MAb 17b may indicate that their binding stabilizes distinct conformational states of gp120. The functional properties of 12p1 suggest that it might be a useful lead for the development of inhibitors of HIV entry.

Type IA and type II DNA topoisomerases are distinguished by their ability to cleave one or two strands, respectively, of a DNA duplex. Both types have been proposed to use an “enzyme-bridging” mechanism, in which a break is formed in a DNA strand and a gap is opened between the broken pieces to allow passage of a second DNA strand or duplex segment. Although the type IA and type II topoisomerase structures appear overall quite different from one another, unexpected similarities between several structural elements suggest that members of the two subfamilies may use comparable mechanisms to bind and cleave DNA.