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

<p>Abstract</p> <p>Background</p> <p>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 <it>E. coli </it>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.</p> <p>Results</p> <p>We have solved the structure of a malate synthase H (MSH) isoform member from <it>Haloferax volcanii </it>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 β8/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.</p> <p>Conclusions</p> <p>The reported structures of MSH from <it>Haloferax volcanii </it>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.</p

Characterization of resistance to a potent D-peptide HIV entry inhibitor.

BACKGROUND: PIE12-trimer is a highly potent D-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (\u3e 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket. RESULTS: Using deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data. CONCLUSIONS: These data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified

Seroprevalence and Risk Factors for Human Herpesvirus 8 Infection, Rural Egypt1

Seroprevalence and Risk Factors for Human Herpesvirus 8 Infection, Rural Egypt, Salivary and nosocomial transmission are possible

A simple device for studying macromolecular crystals under moderate gas pressures (0.1-10 MPa)

A simple device for studying crystalline samples under moderate gas pressure (0.1-10 MPa) has been developed. The device employs a modified Cajon ultra-torr fitting to ensure a gas-tight seal around an X-ray capillary. The cell accommodates standard X-ray capillaries that require no modification. The device is straightforward to utilize and samples can be mounted with routine techniques and pressurized in a matter of seconds. In a subsequent development, a complete purging and pressurization system has been designed and constructed for use on beamline 7-1 at the Stanford Synchrotron Radiation Laboratory. This paper describes the construction of both the pressure cell and the delivery system and presents results of the use of this cell for the preparation of xenon derivatives to be used in phase determination by the multiple isomorphous replacement method

Structure of tropomyosin at 7A resolution

The crystal structure of tropomyosin has been determined by X-ray diffraction analysis at 7A resolution. Tropomyosin is a 400A-long muscle regulatory protein that consists of two parallel 33,000 Dalton alpha helices wound around one another to form a coiled coil and whose amino-acid sequence is characterized by a characteristic heptad repeat pattern. The structure was solved initially at 9A resolution by molecular replacement and refinement of a uniform wire model with a specially designed refinement procedure. Phase information was later derived from a single mercury derivative by single isomorphous replacement (SIR) refinement and used in the construction of an atomic model which was refined at 7A resolution. The model agrees well with the previous low-resolution X-ray structure and with models of tropomyosin in paracrystalline and micro-crystalline forms based on electron microscopy. The overall shape of the molecules, characteristics of the coiled coil and the geometry of interactions of molecules in the crystal are apparent from the structure. The molecules are precipitated by spermine and polymerize head-to-tail to form sheets of nearly parallel filaments, overlapping by about 2/3 of the molecular length in an antiparallel configuration. The relationship of two cysteine residues on each of the molecules was determined unambiguously by solving the structure of a mercury-labeled form of the protein. The shape of the molecule is influenced by local amino-acid sequence variations and crystal packing interactions. The inherent mobility of the molecule in the crystal indicates the importance of considering the flexibility and motions of tropomyosin in models of muscle thin filament regulation and cooperativity. The detailed structure of the head-to-tail overlap region cannot be ascertained from the present model, but will be an important focus of attention for future study. Through amino-acid sequence analysis, an element of quaternary structure, the coiled coil, can be directly predicted. However, tropomyosin is the largest of this class of proteins whose structure has actually been determined by X-ray crystallography