Structural insights into the extracytoplasmic thiamine-binding lipoprotein p37 of Mycoplasma hyorhinis.

The Mycoplasma hyorhinis protein p37 has been implicated in tumorigenic transformation for more than 20 years. Though there are many speculations as to its function, based solely on sequence homology, the issue has remained unresolved. Presented here is the 1.6-A-resolution refined crystal structure of M. hyorhinis p37, renamed the extracytoplasmic thiamine-binding lipoprotein (Cypl). The structure shows thiamine pyrophosphate (TPP) and two calcium ions are bound to Cypl and give the first insights into possible functions of the Cypl-like family of proteins. Sequence alignments of Cypl-like proteins between several different species of mycoplasma show that the thiamine-binding site is likely conserved and structural alignments reveal the similarity of Cypl to various binding proteins. While the experimentally determined function of Cypl remains unknown, the structure shows that the protein is a TPP-binding protein, opening up many avenues for future mechanistic studies and making Cypl a possible target for combating mycoplasma infections and tumorigenic transformation.

Structure determination of the cancer-associated Mycoplasma hyorhinis protein Mh-p37.

The crystal structure of the Mycoplasma hyorhinis protein Mh-p37 has been solved and refined to 1.9 A resolution. This is the first de novo structure to be determined using the recently described heavy-atom reagent [Beck et al. (2008), Acta Cryst. D64, 1179-1182] 5-amino-2,4,6-triiodoisophthalic acid (I3C), which contains three I atoms arranged in an equilateral triangle, by SIRAS methods. Data collection was performed in-house at room temperature. SHELXD and SHELXE were used to determine the I-atom positions and phase the native protein and PHENIX AutoBuild software was used to automatically fit the amino-acid sequence to the electron-density map. The structure was refined using SHELX97 to an R(cryst) of 18.6% and an R(free) of 24.0%. Mh-p37 is an alpha/beta protein with two well defined domains which are separated by a deep cleft. An unanticipated ligand bound in the center of the molecule at the base of the cleft has been modeled as thiamine pyrophosphate or vitamin B(1). Retrospective attempts to solve the crystal structure by Patterson search methods using either isomorphous or anomalous differences failed. Additionally, attempts to use proteins with the highest structural homology in the Protein Data Bank to phase the data by molecular replacement were unsuccessful, most likely in hindsight because of their poor structural agreement. Therefore, the I3C reagent offers an alternative, quick and inexpensive method for in-house phasing of de novo structures where other methods may not be successful.

An unstable head-rod junction may promote folding into the compact off-state conformation of regulated myosins.

The N-terminal region of myosin's rod-like subfragment 2 (S2) joins the two heads of this dimeric molecule and is key to its function. Previously, a crystal structure of this predominantly coiled-coil region was determined for a short fragment (51 residues plus a leucine zipper) of the scallop striated muscle myosin isoform. In that study, the N-terminal 10-14 residues were found to be disordered. We have now determined the structure of the same scallop peptide in three additional crystal environments. In each of two of these structures, improved order has allowed visualization of the entire N-terminus in one chain of the dimeric peptide. We have also compared the melting temperatures of this scallop S2 peptide with those of analogous peptides from three other isoforms. Taken together, these experiments, along with examination of sequences, point to a diminished stability of the N-terminal region of S2 in regulated myosins, compared with those myosins whose regulation is thin filament linked. It seems plain that this isoform-specific instability promotes the off-state conformation of the heads in regulated myosins. We also discuss how myosin isoforms with varied thermal stabilities share the basic capacity to transmit force efficiently in order to produce contraction in their on states.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

Crystal structure of nitrated human manganese superoxide dismutase: mechanism of inactivation.

A cellular consequence of the reaction of superoxide and nitric oxide is enhanced peroxynitrite levels. Reaction of peroxynitrite with manganese superoxide dismutase (MnSOD) causes nitration of the active-site residue Tyr34 and nearly complete inhibition of catalysis. We report the crystal structures at 2.4 A resolution of human MnSOD nitrated by peroxynitrite and the unmodified MnSOD. A comparison of these structures showed no significant conformational changes of active-site residues or solvent displacement. The side chain of 3-nitrotyrosine 34 had a single conformation that extended toward the manganese with O1 of the nitro group within hydrogen-bonding distance (3.1 A) of Nepsilon2 of the second-shell ligand Gln143. Also, nitration of Tyr34 caused a weakening, as evidenced by the lengthening, of a hydrogen bond between its phenolic OH and Gln143, part of an extensive hydrogen-bond network in the active site. Inhibition of catalysis can be attributed to a steric effect of 3-nitrotyrosine 34 that impedes substrate access and binding, and alteration of the hydrogen-bond network that supports proton transfer in catalysis. It is also possible that an electrostatic effect of the nitro group has altered the finely tuned redox potential necessary for efficient catalysis, although the redox potential of nitrated MnSOD has not been measured.

p37 Induces tumor invasiveness.

Previous studies have shown a statistically significant correlation between human carcinomas and monoclonal antibody detection of a Mycoplasma hyorhinis-encoded protein known as p37. A potential mechanism of p37 is that it might promote invasion and metastasis. Recombinant p37 enhanced the invasiveness of two prostate carcinoma and two melanoma cell lines in a dose-dependent manner in vitro, but did not have a significant effect on tumor cell growth. Furthermore, the increased binding to cell surfaces and the enhanced invasive potential of cancer cells from exposure to p37 could be completely reversed by preincubation of the cancer cells with an anti-p37 monoclonal antibody. Sequence comparisons, followed by three-dimensional molecular modeling, revealed a region of similarity between p37 and influenza hemagglutinin A, a sialic acid-binding protein that plays a critical role in viral entry. Binding of p37 to prostate carcinoma cells was found to be at least partially sialic acid dependent because neuraminidase treatment decreased this binding. Taken together, these observations suggest that M. hyorhinis can infect humans and may facilitate tumor invasiveness via p37. These results further suggest that p37 may be a molecular target for cancer therapy.

Comparing the accumulation of active- and nonactive-site mutations in the HIV-1 protease.

Protease inhibitor resistance still poses one of the greatest challenges in treating HIV. To better design inhibitors able to target resistant proteases, a deeper understanding is needed of the effects of accumulating mutations and the contributions of active- and nonactive-site mutations to the resistance. We have engineered a series of variants containing the nonactive-site mutations M46I and I54V and the active-site mutation I84V. These mutations were added to a protease clone (V6) isolated from a pediatric patient on ritonavir therapy. This variant possessed the ritonavir-resistance-associated mutations in the active-site (V32I and V82A) and nonactive-site mutations (K20R, L33F, M36I, L63P, A71V, and L90M). The I84V mutation had the greatest effect on decreasing catalytic efficiency, 10-fold when compared to the pretherapy clone LAI. The decrease in catalytic efficiency was partially recovered by the addition of mutations M46I and I54V. The M46I and I54V were just as effective at decreasing inhibitor binding as the I84V mutation when compared to V6 and LAI. The V6(54/84) variant showed over 1000-fold decrease in inhibitor-binding strength to ritonavir, indinavir, and nelfinavir when compared to LAI and V6. Crystal-structure analysis of the V6(54/84) variant bound to ritonavir and indinavir shows structural changes in the 80's loops and active site, which lead to an enlarged binding cavity when compared to pretherapy structures in the Protein Data Bank. Structural changes are also seen in the 10's and 30's loops, which suggest possible changes in the dynamics of flap opening and closing.

Structural determination of a partially hemihedrally twinned actin crystal.

An orthorhombic actin crystal (space group P2(1)2(1)2(1), unit-cell parameters a = 101.6, b = 103.0, c = 127.0 angstroms) was converted into a partially hemihedrally twinned tetragonal crystal (space group P4(3), unit-cell parameters a = b = 101.5, c = 104.2 angstroms) by induced condensation. This condensation (decrease in the c axis) was caused by the flash-freezing of the crystal, with 30% PEG 400 as a cryoprotectant, prior to data collection. Diffraction data for the twinned tetragonal crystal were collected at 100 K to 3.0 angstroms resolution (99.8% completeness with an Rsym of 8.1%) using synchrotron radiation. The hemihedral twinning of the data was observed by self-rotation function analysis and was determined to have a partial twin fraction of 0.376 from intensity statistics. The structure, with two actin molecules in the crystallographic asymmetric unit, was determined by molecular-replacement methods and refined to an R factor of 0.193. As a consequence of the crystal lattice transformation from the orthorhombic P2(1)2(1)2(1) to the tetragonal P4(3) space group, actin-actin contacts were rearranged and an inter-actin dimer disulfide bond (Cys374) observed in the orthorhombic crystal form was broken in the tetragonal crystal form.

Actin crystal dynamics: structural implications for F-actin nucleation, polymerization, and branching mediated by the anti-parallel dimer.

Actin filament nucleation, polymerization, and branching are crucial steps in many forms of cell motility, cell shape, and intracellular organelle movements in a wide range of organisms. Previous biochemical data suggests that an anti-parallel actin dimer can incorporate itself into growing filamentous actin (F-actin) and has a role in branching. Furthermore, it is a widespread belief that nucleation is spawned from an actin trimer complex. Here we present the structures of actin dimers and trimers in two tetragonal crystal systems P4(3)2(1)2 and P4(3). Both crystal systems formed by an induced condensation transformation of a previously reported orthorhombic crystal system P2(1)2(1)2(1). Comparison between the three crystal systems demonstrates the dynamics and flexibility of actin-actin interactions. The dimer and trimer actin rearrangements observed between the three crystal systems may provide insight to in vivo actin-actin interactions that occur during the nucleation, polymerization, and branching of F-actin.

Crystallization and preliminary X-ray analysis of the tumor metastasis factor p37.

P37, an outer-membrane bacterial protein from Mycoplasma hyorhinis, is a molecule whose presence on the surface of many tumor cells correlates highly with increased neoplastic invasivity and metastasis. P37 was overexpressed in Escherichia coli, purified by affinity chromatography and crystallized. Useful single crystals for X-ray diffraction structural studies have been grown by oil-immersion methods from a solution of 40% PEG 4000, 0.1 M ammonium bromide in a 0.1 M citrate buffer at pH 4.0. X-ray diffraction data were collected at the F2 beamline at CHESS with a crystal-to-CCD detector distance of 150 mm, collecting 1 degrees oscillation slices with an exposure time of 30 s per frame. A 212 degrees sweep of data (99.8% completeness) were collected from a single crystal under cryoconditions, with a maximal useful diffraction pattern to 1.8 A resolution. The crystals are shown to be monoclinic and have been assigned to space group P2(1), with unit-cell parameters a = 50.02, b = 67.26, c = 59.89 A, beta = 108.29 degrees and a scaling R(sym) of 0.076 for 34,882 unique reflections. Packing considerations indicate that there is one molecule per asymmetric unit. It is expected that in the near future the structure of p37 will be obtained using phases from traditional heavy-atom isomorphous replacement and/or halide-soak methods. Elucidation of the structure of p37 may be paramount to producing new antibody-based anticancer therapeutic agents.