Barbara A.L. Owen

The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair.

The Mre11 complex (Mre11–Rad50–Nbs1) is central to chromosomal maintenance and functions in homologous recombination, telomere maintenance and sister chromatid association. These functions all imply that the linked binding of two DNA substrates occurs, although the molecular basis for this process remains unknown. Here we present a 2.2 Å crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn2+ ion. Biochemical, X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 Å. This suggests a function for the long insertion in the Rad50 ABC-ATPase domain. The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homologous end-joining.

(CAG) n -hairpin DNA binds to Msh2–Msh3 and changes properties of mismatch recognition

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2–Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2–Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2–Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2–Msh3 function depend on the presence of A·A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2–Msh3–CAG hairpin complex that could misdirect the DNA repair process.

Crystal Structure and Activity of Human p23, a Heat Shock Protein 90 Co-chaperone

p23 is a co-chaperone for the heat shock protein, hsp90. This protein binds hsp90 and participates in the folding of a number of cell regulatory proteins, but its activities are still unclear. We have solved a crystal structure of human p23 lacking 35 residues at the COOH terminus. The structure reveals a disulfide-linked dimer with each subunit containing eight β-strands in a compact antiparallel β-sandwich fold. In solution, however, p23 is primarily monomeric and the dimer appears to be a minor component. Conserved residues are clustered on one face of the monomer and define a putative surface region and binding pocket for interaction(s) with hsp90 or protein substrates. p23 contains a COOH-terminal tail that is apparently less structured and is unresolved in the crystal structure. This tail is not needed for the binding of p23 to hsp90 or to complexes with the progesterone receptor. However, the tail is necessary for optimum active chaperoning of the progesterone receptor, as well as the passive chaperoning activity of p23 in assays measuring inhibition of heat-induced protein aggregation.

Altered dimer interface decreases stability in an amyloidogenic protein.

Amyloidoses are devastating and currently incurable diseases in which the process of amyloid formation causes fatal cellular and organ damage. The molecular mechanisms underlying amyloidoses are not well known. In this study, we address the structural basis of immunoglobulin light chain amyloidosis, which results from deposition of light chains produced by clonal plasma cells. We compare light chain amyloidosis protein AL-09 to its wild-type counterpart, the κI O18/O8 light chain germline. Crystallographic studies indicate that both proteins form dimers. However, AL-09 has an altered dimer interface that is rotated 90° from the κI O18/O8 dimer interface. The three non-conservative mutations in AL-09 are located within the dimer interface, consistent with their role in the decreased stability of this amyloidogenic protein. Moreover, AL-09 forms amyloid fibrils more quickly than κI O18/O8 in vitro. These results support the notion that the increased stability of the monomer and delayed fibril formation, together with a properly formed dimer, may be protective against amyloidogenesis. This could open a new direction into rational drug design for amyloidogenic proteins.

Bacteriophage φ29 scaffolding protein gp7 before and after prohead assembly

Three-dimensional structures of the double-stranded DNA bacteriophage φ29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 Å, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.

Quantification of hypercoagulable state after blunt trauma: Microparticle and thrombin generation are increased relative to injury severity, while standard markers are not

BACKGROUND Major trauma is an independent risk factor for developing venous thromboembolism. While increases in thrombin generation and/or procoagulant microparticles have been detected in other patient groups at greater risk for venous thromboembolism, such as cancer or coronary artery disease, this association has yet to be documented in trauma patients. This pilot study was designed to characterize and quantify thrombin generation and plasma microparticles in individuals early after traumatic injury. METHODS Blood was collected in the trauma bay from 52 blunt injured patients (cases) and 19 uninjured outpatients (controls) and processed to platelet poor plasma to allow for (1) isolation of microparticles for identification and quantification by flow cytometry, and (2) in vitro thrombin generation as measured by calibrated automatic thrombography. Data collected are expressed as either mean ± standard deviation or median with interquartile range. RESULTS Among the cases, which included 39 men and 13 women (age, 40 ± 17 years), the injury severity score was 13 ± 11, the international normalized ratio was 1.0 ± 0.1, the thromboplastin time was 25 ± 3 seconds, and platelet count was 238 ± 62 (thousands). The numbers of total (cell type not specified) procoagulant microparticles, as measured by Annexin V staining, were increased compared to nontrauma controls (541 ± 139/μL and 155 ± 148/μL, respectively; P < .001). There was no significant difference in the amount of thrombin generated in trauma patients compared to controls; however, peak thrombin was correlated to injury severity (Spearman correlation coefficient R, 0.35; P = .02). CONCLUSION Patients with blunt trauma have greater numbers of circulating procoagulant microparticles and increased in vitro thrombin generation. Future studies to characterize the cell-specific profiles of microparticles and changes in thrombin generation kinetics after traumatic injury will determine whether microparticles contribute to the hypercoagulable state observed after injury.

A single mutation promotes amyloidogenicity through a highly promiscuous dimer interface.

Light chain amyloidosis is a devastating protein misfolding disease characterized by the accumulation of amyloid fibrils that causes tissue damage and organ failure. These fibrils are composed of monoclonal light chain protein secreted from an abnormal proliferation of bone marrow plasma cells. We previously reported that amyloidogenic light chain protein AL-09 adopts an altered dimer while its germline protein (kappaI O18/O8) forms a canonical dimer observed in other light chain crystal structures. In solution, conformational heterogeneity obscures all NMR signals at the AL-09 and kappaI O18/O8 dimer interfaces, so we solved the nuclear magnetic resonance structure of two related mutants. AL-09 H87Y adopts the normal dimer interface, but the kappaI Y87H solution structure presents an altered interface rotated 180 degrees relative to the canonical dimer interface and 90 degrees from the AL-09 arrangement. Our results suggest that promiscuity in the light chain dimer interface may promote new intermolecular contacts that may contribute to amyloid fibril structure.

Procoagulant activity, but not number, of microparticles increases with age and in individuals after a single venous thromboembolism

The Calibrated Automated Thrombogram (CAT), a plate-based assay that measures thrombin generation and inhibition in plasma samples, is modified to measure the procoagulant activity of phospholipid associated with plasma microparticles (MP). The assay uses a tissue factor trigger without addition of 4 μM exogenous phospholipid (PL) used in the standard CAT. Calibrated with 4:1 posphatidylcholine- phosphatidylserine (PCPS) liposomes, the assay defines a median of 40 nM procoagulant phospholipid (PPL) equivalents in plasma containing MPs from 50 normal donors, with a range from 20 - 200 nM. Like the standard CAT, the modified assay detected no difference in plasma from 36 individuals with a history of a single episode of venous thromboembolism. However the male cases had double the PPL activity, as measured by rate of thrombin generation, of females; and there was a significant correlation among cases of increased thrombin generation with age. In contrast, there were no gender disparities or age correlations among control plasmas. The findings suggest that procoagulant activity of plasma microparticles, facilitated by a simplified, one-stage plate-based assay, offer a promising avenue of investigation of mechanisms and management of venous thromboembolic disorders.

Thermal Stability of MHC Class I-β2-Microglobulin Peptide Complexes in the Endoplasmic Reticulum Is Determined by the Peptide Occupancy of the Transporter Associated with Antigen Processing Complex

Once MHC class I heavy chain binds β2-microglobulin (β2m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated Kbβ2m complexes within microsomes (TAP+/TAPasin+) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of Kb-β2m-peptide complexes depends on the timing of peptide binding to Kbβ2m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in Kbβ2m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of Kbβ2m-peptide complexes.

Corrigendum: (CAG) n -hairpin DNA binds to Msh2–Msh3 and changes properties of mismatch recognition

Nat. Struct. Mol. Biol. 12, 663–653 (2005). The manuscript contained an error in the name of one of the authors. Maciej Gajec was misspelled Maciez Gajek. The correct author list should read: Barbara A L Owen, Zungyoon Zang, Maoyi Lai, Maciej Gajec, John D Badger II, Jeffrey J Hayes, Winfried Edelmann, Raju Kucherlapati, Teresa M Wilson & Cynthia T McMurray.