A conformational switch controlling HIV-1 morphogenesis.

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) proceeds in two steps. Initially, an immature virus with a spherical capsid shell consisting of uncleaved Gag polyproteins is formed. Extracellular proteolytic maturation causes rearrangement of the inner virion structure, leading to the conical capsid of the infectious virus. Using an in vitro assembly system, we show that the same HIV-1 Gag-derived protein can form spherical particles, virtually indistinguishable from immature HIV-1 capsids, as well as tubular or conical particles, resembling the mature core. The assembly phenotype could be correlated with differential binding of the protein to monoclonal antibodies recognizing epitopes in the HIV-1 capsid protein (CA), suggesting distinct conformations of this domain. Only tubular and conical particles were observed when the protein lacked spacer peptide SP1 at the C-terminus of CA, indicating that SP1 may act as a molecular switch, whose presence determines spherical capsid formation, while its cleavage leads to maturation.

In vitro assembly properties of wild-type and cyclophilin-binding defective human immunodeficiency virus capsid proteins in the presence and absence of cyclophilin A.

The cellular protein cyclophilin A (CypA) binds specifically to the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein and is incorporated into HIV-1 particles at a molar ratio of 1:10 (CypA/CA). Structural analysis of a CA-CypA complex suggested that CypA may destabilize interactions in the viral capsid and thus promote uncoating. We analyzed the influence of CypA on the in vitro assembly properties of wild-type (WT) CA and derivatives containing substitutions of Gly89 in the Cyp-binding loop. All variant proteins were significantly impaired in CypA binding. In the presence of CypA at a molar ratio of 1:10 (CypA/CA), WT CA assembled into hollow cylinders that were similar to those observed in the absence of CypA but slightly longer. Higher CypA concentrations inhibited cylinder formation. Variant CA proteins G89L and G89F yielded similar cylinders as the WT protein but were significantly more resistant to CypA. Cryoelectron microscopic analysis of WT cylinders assembled in the presence of CypA revealed direct binding of CypA to the outer surface. Electron diffraction patterns generated from these cylinders indicated that CypA causes local disorder. The addition of CypA to preassembled cylinders had little effect, however, and cylinders were only disrupted when incubated with a threefold molar excess of CypA for several hours. These results suggest that CypA does not efficiently destabilize CA interactions at the molar ratio observed in the virion and therefore is unlikely to serve as an uncoating factor.

Cyclophilin A incorporation is not required for human immunodeficiency virus type 1 particle maturation and does not destabilize the mature capsid.

The cellular protein cyclophilin A (CypA) is packaged into human immunodeficiency virus type 1 (HIV-1) virions through a specific interaction with the capsid (CA) domain of the Gag polyprotein. CypA is important for infectivity, but its role in viral replication is currently unknown. Previous reports suggested that CypA promotes uncoating or enhances maturation. We analyzed the morphology and capsid stability of HIV-1 variants defective in CypA binding and of virus grown in the presence of cyclosporin. Both cyclosporin treatment and alteration of Gly89 or Pro90 in the CypA-binding site of CA caused a 5- to 20-fold decrease in CypA incorporation. Virus produced from cyclosporin-treated cells and variants G89V and G89A were 10- to 100-fold less infectious but exhibited normal virion morphologies with regular cone-shaped capsids. Irregular capsid morphologies and lower infectivities were observed for some other variants in the CypA-binding region. Decreased CypA incorporation did not reduce the kinetics of intracellular polyprotein processing or of virus release. No increase in immature particles was observed. These results suggest that CypA does not promote virion maturation. Furthermore, detergent stripping of virus particles with various CypA contents revealed no difference in capsid stability. Based on these results and those reported in the accompanying paper, it appears likely that CypA also is not an uncoating factor. Alternative models for CypA function are discussed.

Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima: catalytic, spectral and thermodynamic properties.

Recombinant phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima (TmPGK) has been expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity applying heat incubation of the crude extract at 80 degreesC, ion exchange chromatography and gel filtration. The biochemical, catalytic and spectral properties were compared with those of the natural enzyme and found to be identical. As shown by SDS-PAGE, ultracentrifugal analysis and gel filtration chromatography, the enzyme is a 43 kDa monomer. At neutral pH, the guanidinium chloride (GdmCl) and temperature-induced denaturation transitions reveal two-state behaviour with high cooperativity. As taken from the temperature dependence of the free energy of unfolding at zero GdmCl concentration and pH 7, optimum stability is observed at approximately 30 degreesC. The difference in the free energies of stabilization for the enzymes from yeast and Thermotoga amounts to Delta DeltaG=85 kJ/mol. The extrapolated temperatures of cold and heat-denaturation are about -10 and +85 degreesC. This indicates that the stability profile of TmPGK is shifted to higher free energy values and broadened over a wider temperature range, compared to that observed for PGKs from mesophiles or moderately thermophiles. In order to achieve cold or heat-denaturation, GdmCl concentrations of approximately 1.8 or approximately 0.9 M are required. Due to a kinetic intermediate on the pathway of cold denaturation, equilibration in the transition range takes exceedingly long.

Crystallographic analysis of phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima.

Phosphoglycerate kinase from the hyperthermophilic bacterium Thermotoga maritima has been co-crystallized with its substrate 3-phosphoglycerate and the ATP analogue AMP-PNP using the vapour diffusion method. Crystals were obtained from a solution containing polyethylene glycol (MW 3000/8000) as precipitating agent. A complete diffraction data set from orthorhombic crystals was collected up to 2.0 A resolution. The TmPGK crystallizes in the space group P2(1)2(1)2 (cell dimensions: a = 62.0 A, b = 76.9 A, c = 87.5 A) with one molecule in the asymmetric unit. The structure was solved by Patterson search methods using Bacillus stearothermophilus PGK as a search model and was refined to a crystallographic R factor of 22.0%. Compared to the enzyme from B. stearothermophilus, horse, pig and yeast, the Thermotoga enzyme exhibits a drastically reduced interdomain angle, similar to the one reported for PGK from Trypanosoma brucei. Here we present crystallographic data of the first high-resolution structure of a PGK in largely closed conformation, complexed with the two products of the catalyzed reaction, and, at the same time, the first PGK structure from a hyperthermophilic organism.

Closed structure of phosphoglycerate kinase from Thermotoga maritima reveals the catalytic mechanism and determinants of thermal stability.

BACKGROUND: Phosphoglycerate kinase (PGK) is essential in most living cells both for ATP generation in the glycolytic pathway of aerobes and for fermentation in anaerobes. In addition, in many plants the enzyme is involved in carbon fixation. Like other kinases, PGK folds into two distinct domains, which undergo a large hinge-bending motion upon catalysis. The monomeric 45 kDa enzyme catalyzes the transfer of the C1-phosphoryl group from 1, 3-bisphosphoglycerate to ADP to form 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. For decades, the conformation of the enzyme during catalysis has been enigmatic. The crystal structure of PGK from the hyperthermophilic organism Thermotoga maritima (TmPGK) represents the first structure of an extremely thermostable PGK. It adds to a series of four known crystal structures of PGKs from mesophilic via moderately thermophilic to a hyperthermophilic organism, allowing a detailed analysis of possible structural determinants of thermostability. RESULTS: The crystal structure of TmPGK was determined to 2.0 A resolution, as a ternary complex with the product 3-phosphoglycerate and the product analogue AMP-PNP (adenylyl-imido diphosphate). The complex crystallizes in a closed conformation with a drastically reduced inter-domain angle and a distance between the two bound ligands of 4.4 A, presumably representing the active conformation of the enzyme. The structure provides new details of the catalytic mechanism. An inter-domain salt bridge between residues Arg62 and Asp200 forms a strap to hold the two domains in the closed state. We identify Lys197 as a residue involved in stabilization of the transition state phosphoryl group, and so term it the 'phosphoryl gripper'. CONCLUSIONS: The hinge-bending motion of the two domains upon closure of the structure, as seen in the Trypanosoma PGK structure, is confirmed. This closed conformation obviously occurs after binding of both substrates and is locked by the Arg62-Asp200 salt bridge. Re-orientations in the conserved active-site loop region around Thr374 also bring both domains into direct contact in the core region of the former inter-domain cleft, to form the complete catalytic site. Comparison of extremely thermostable TmPGK with less thermostable homologues reveals that its increased rigidity is achieved by a raised number of intramolecular interactions, such as an increased number of ion pairs and additional stabilization of alpha helix and loop regions. The covalent fusion with triosephosphate isomerase might represent an additional stabilization strategy.