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. 2009 May;83(9):4395-403.
doi: 10.1128/JVI.02352-08. Epub 2009 Feb 25.

Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus

Robert A Love  1 , Oleg Brodsky, Michael J Hickey, Peter A Wells, Ciarán N Cronin
Affiliations

Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus

Robert A Love et al. J Virol. 2009 May.

Abstract

A new protein expression vector design utilizing an N-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis C virus NS5A sequence has resulted in a more straightforward purification method and improved yields of purified NS5A domain I protein. High-resolution diffracting crystals of NS5A domain I (amino acids 33 to 202) [NS5A(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is organized differently from that published previously (T. L. Tellinghuisen, J. Marcotrigiano, and C. M. Rice, Nature 435:374-379, 2005) yet is consistent with a membrane association model for NS5A. The monomer-monomer interface of NS5A(33-202) features an extensive buried surface area involving the most-highly conserved face of each monomer. The two alternate structural forms of domain I now available may be indicative of the multiple roles emerging for NS5A in viral RNA replication and viral particle assembly.

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Figures

FIG. 1.
FIG. 1.
Overall structure of the NS5A(33-202) dimer. (A and B) Ribbon (A) and molecular surface (B) representations, with monomer long axes vertical, N termini at the top, C termini at the bottom, and Zn atoms. IA and IB show subdomains of each monomer. (C and D) Views similar to those in panels A and B but looking down the long axes of the monomers.
FIG. 2.
FIG. 2.
Comparison of the two known structures of NS5A domain I. (A) Side view. (B) Top view. The dimer of NS5A(33-202) (blue/magenta) is aligned with that from the previously reported NS5A(25-215) dimer (yellow/orange) following superposition of one monomer from each structure. Transformation between these two configurations requires complete separation of the monomers along with twisting of the long axes.
FIG. 3.
FIG. 3.
Electrostatic surface potential for the NS5A(33-202) dimer, with blue denoting positive and red negative values. The N termini of both monomers are located at the top, and the C termini are located at the bottom. For reference, the location of the second monomer in the NS5A(25-215) dimer is included as a stick representation, with overall orientation similar to that depicted in Fig. 2A.
FIG. 4.
FIG. 4.
Dimer interface of NS5A(33-202) as seen looking through monomer B (yellow ribbon) onto monomer A (blue surface). Residues of monomer A in contact with monomer B are colored either magenta or green. Magenta signifies amino acid conservation above 95% based on 35 sequences spanning genotypes 1 to 6 from the Los Alamos database (20). Water molecules at the interface are shown as red spheres, and the trapped glycerol is an orange stick model. Intermolecular main-chain-main-chain H bonds formed between Gly 96 N and Ala 92 O are colored and labeled separately.
FIG. 5.
FIG. 5.
Key intermolecular interactions at the dimer interface in the NS5A(33-202) structure. (A) Intermolecular hydrogen bonds include highly conserved Arg 112 (occupied rarely by Lys) paired with invariant Glu 148 and main-chain atom Ala 92 O interacting with Gly 96 N. Invariant Arg 160 forms intermolecular contact with Ala 92 O, along with an intramolecular hydrogen bond to Gly 96. (B) Aromatic side chains at the interface. At position 93 (least buried), Tyr is predominant, although His and Thr sometimes arise. At position 161, Phe and Tyr are found about equally but His occurs on occasion. At position 149 (most buried), Phe is predominant, with the rare exception of Leu. Monomer A has a surface with highlighted colors for residues denoted “a.” Monomer B is displayed as a ribbon with stick sidechains for residues denoted “b.” Distances shown are in angstroms.
FIG. 6.
FIG. 6.
Small molecules observed in crystals of NS5A(33-202). (A) Glycerol molecule (yellow) found at the dimer interface, with hydrogen bond distances in Å. Glycerol was present in the protein and crystallization buffer, and this binding is probably a fortuitous event rather than a determinant of dimer formation. Red spheres show ordered water molecules at the interface. (B) Maltose detergent molecule (yellow) bound to surface of monomer A (blue surface), with its second sugar ring disordered. Also shown is monomer B of the dimer (magenta surface), along with the expected location for a second monomer in the NS5A(25-215) configuration (orange surface). The detergent is distant from either dimer interface and appears to facilitate contacts between an NS5A(33-202) dimer and a symmetry-related dimer (green ribbon).
FIG. 7.
FIG. 7.
Theoretical assembly of NS5A domain I proteins based on simultaneous recruitment of the two monomer-monomer interfaces, resulting in a superhelical array. (A) Side view. In this depiction, NS5A(25-215) dimers are distinguished by separate colors and are joined by means of the interface identified in the NS5A(33-202) dimer. One full “turn” of the superhelix consists, for example, of dimers colored green, orange, magenta, and cyan, and then green again. The two C termini of each dimer are colored red and project outward, implying that domains II and III of NS5A could be accommodated sterically. The location of each emerging N-terminal residue 36 is colored blue. (B) View down long axis of superhelix, with only four dimers displayed for clarity, colored green (nearest), orange, magenta, and cyan (farthest). The two N termini of each dimer (residue 36) are indicated by a sphere with color matching the dimer. The C termini are in red. This arrangement suggests that the N-terminal amphipathic helices (not modeled) would be directed inward toward the assembly's central axis, where they might interact with one another. Any membrane associated with these amphipathic helices could not adopt a planar geometry but instead would be highly twisted. Currently there is no evidence for such an assembly in the cellular environment, but this polymerization may explain the properties of isolated NS5A in solution, which include a highly heterogeneous oligomeric state.
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