Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G.

BACKGROUND: Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. RESULTS: We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a ß8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. CONCLUSIONS: The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H isoform, the catalytic mechanism is conserved. Sequence analysis in light of the structure indicates that additional members of isoform H likely exist in the databases but have been misannotated.

Structure of the antiviral assembly inhibitor CAP-1 complex with the HIV-1 CA protein.

The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N'-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.

Implications for viral capsid assembly from crystal structures of HIV-1 Gag(1-278) and CA(N)(133-278).

Gag, the major structural protein of retroviruses such as HIV-1, comprises a series of domains connected by flexible linkers. These domains drive viral assembly by mediating multiple interactions between adjacent Gag molecules and by binding to viral genomic RNA and host cell membranes. Upon viral budding, Gag is processed by the viral protease to liberate distinct domains as separate proteins. The first two regions of Gag are MA, a membrane-binding module, and CA, which is a two-domain protein that makes important Gag-Gag interactions, forms the cone-shaped outer shell of the core (the capsid) in the mature HIV-1 particle, and makes an important interaction with the cellular protein cyclophilin A (CypA). Here, we report crystal structures of the mature CA N-terminal domain (CA(N)(133-278)) and a MA-CA(N) fusion (Gag(1-278)) at resolutions/R(free) values of 1.9 A/25.7% and 2.2 A/25.8%, respectively. Consistent with earlier studies, a comparison of these structures indicates that processing at the MA-CA junction causes CA to adopt an N-terminal beta-hairpin conformation that seems to be required for capsid morphology and viral infectivity. In contrast with an NMR study (Tang, C., et al. (2002) Nat. Struct. Biol. 9, 537-543), structural overlap reveals only small relative displacements for helix 6, which is located between the beta-hairpin and the CypA-binding loop. These observations argue against the proposal that CypA binding is coupled with beta-hairpin formation and support an earlier surface plasmon resonance study (Yoo, S., et al. (1997) J. Mol. Biol. 269, 780-795), which concluded that beta-hairpin formation and CypA-binding are energetically independent events.

Structural insights into the catalytic mechanism of cyclophilin A.

Cyclophilins constitute a ubiquitous protein family whose functions include protein folding, transport and signaling. They possess both sequence-specific binding and proline cis-trans isomerase activities, as exemplified by the interaction between cyclophilin A (CypA) and the HIV-1 CA protein. Here, we report crystal structures of CypA in complex with HIV-1 CA protein variants that bind preferentially with the substrate proline residue in either the cis or the trans conformation. Cis- and trans-Pro substrates are accommodated within the enzyme active site by rearrangement of their N-terminal residues and with minimal distortions in the path of the main chain. CypA Arg55 guanidinium group probably facilitates catalysis by anchoring the substrate proline oxygen and stabilizing sp3 hybridization of the proline nitrogen in the transition state.