Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs.

The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 "nonstructural" proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 A as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular beta-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the "gag-knuckle fold group" of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

Coiled coils at the edge of configurational heterogeneity. Structural analyses of parallel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a single solvent-exposed amino acid substitution.

A detailed understanding of the mechanisms by which particular amino acid sequences can give rise to more than one folded structure, such as for proteins that undergo large conformational changes or misfolding, is a long-standing objective of protein chemistry. Here, we describe the crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and further describe the parallel or antiparallel crystal structures of several related peptide sequences; the antiparallel tetrameric assemblies represent the first crystal structures of GCN4-derived peptides exhibiting such a configuration. Intriguingly, substitution of a single solvent-exposed residue enabled the parallel coiled-coil tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for subtle sequence changes to have important structural consequences. We present a structural analysis of the small changes to the helix register and side-chain conformations that accommodate the two configurations and have supplemented these results using solution studies and a molecular dynamics energetic analysis using a replica exchange methodology. Considering the previous examples of structural nonspecificity in coiled-coil peptides, the findings reported here not only emphasize the predisposition of the coiled-coil motif to adopt multiple configurations but also call attention to the associated risk that observed crytstal structures may not represent the only (or even the major) species present in solution.

Structure-based engineering of internal cavities in coiled-coil peptides.

Cavities and clefts are frequently important sites of interaction between natural enzymes or receptors and their corresponding substrate or ligand molecules and exemplify the types of molecular surfaces that would facilitate engineering of artificial catalysts and receptors. Even so, structural characterizations of designed cavities are rare. To address this issue, we performed a systematic study of the structural effects of single-amino acid substitutions within the hydrophobic cores of tetrameric coiled-coil peptides. Peptides containing single glycine, serine, alanine, or threonine amino acid substitutions at the buried L9, L16, L23, and I26 hydrophobic core positions of a GCN4-based sequence were synthesized and studied by solution-phase and crystallographic techniques. All peptides adopt the expected tetrameric state and contain tunnels or internal cavities ranging in size from 80 to 370 A(3). Two closely related sequences containing an L16G substitution, one of which adopts an antiparallel configuration and one of which adopts a parallel configuration, illustrate that cavities of different volumes and shapes can be engineered from identical core substitutions. Finally, we demonstrate that two of the peptides (L9G and L9A) bind the small molecule iodobenzene when present during crystallization, leaving the general peptide quaternary structure intact but altering the local peptide conformation and certain superhelical parameters. These high-resolution descriptions of varied molecular surfaces within solvent-occluded internal cavities illustrate the breadth of design space available in even closely related peptides and offer valuable models for the engineering of de novo helical proteins.

In situ data collection and structure refinement from microcapillary protein crystallization.

In situ X-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 Å resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained from in situ diffraction of crystals grown in microcapillaries.

Heterocyclic peptide backbone modifications in an alpha-helical coiled coil.

In this paper, we present 1,2,3-triazole epsilon2-amino acids incorporated as a dipeptide surrogate at three positions in the sequence of a known alpha-helical coiled coil. Biophysical characterization indicates that the modified peptides retain much of the helical structure of the parent sequence, and that the thermodynamic stability of the coiled coil depends on the position of the incorporation of the epsilon-residue. Crystal structures obtained for each peptide give insight into the chemical behavior and conformational preferences of the non-natural amino acid and show that the triazole ring can participate in the backbone hydrogen bonding of the alpha-helix as well as template an interhelical crossing between chains in the bundle.