The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.

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Hydrogen peroxide is a cell signaling agent that inactivates protein tyrosine phosphatases (PTPs) via oxidation of their catalytic cysteine residue. PTPs are inactivated rapidly during H2O2-mediated cellular signal transduction processes but, paradoxically, hydrogen peroxide is a rather sluggish PTP inactivator in vitro. Here we present evidence that the biological buffer, bicarbonate/CO2, potentiates the ability of H2O2 to inactivate PTPs. The results of biochemical experiments and high resolution crystallographic analysis are consistent with a mechanism involving oxidation of the catalytic cysteine residue by peroxymonocarbonate generated via the reaction of H2O2 with HCO3 −/CO2.

ObjectiveTo define a minimum Standard Set of outcome measures and case‐mix factors for monitoring, comparing, and improving health care for patients with clinically diagnosed hip or knee osteoarthritis (OA), with a focus on defining the outcomes that matter most to patients.MethodsAn international working group of patients, arthroplasty register experts, orthopedic surgeons, primary care physicians, rheumatologists, and physiotherapists representing 10 countries was assembled to review existing literature and practices for assessing outcomes of pharmacologic and nonpharmacologic OA therapies, including surgery. A series of 8 teleconferences, incorporating a modified Delphi process, were held to reach consensus.ResultsThe working group reached consensus on a concise set of outcome measures to evaluate patients’ joint pain, physical functioning, health‐related quality of life, work status, mortality, reoperations, readmissions, and overall satisfaction with treatment result. To support analysis of these outcome measures, pertinent baseline characteristics and risk factor metrics were defined. Annual outcome measurement is recommended for all patients.ConclusionWe have defined a Standard Set of outcome measures for monitoring the care of people with clinically diagnosed hip or knee OA that is appropriate for use across all treatment and care settings. We believe this Standard Set provides meaningful, comparable, and easy to interpret measures ready to implement in clinics and/or registries globally. We view this set as an initial step that, when combined with cost data, will facilitate value‐based health care improvements in the treatment of hip and knee OA.

When expressed as part of a glutathione S-transferase fusion protein the NH2-terminal domain of the lymphocyte cell adhesion molecule CD2 is shown to adopt two different folds. The immunoglobulin superfamily structure of the major (85%) monomeric component has previously been determined by both x-ray crystallography and NMR spectroscopy. We now describe the structure of a second, dimeric, form present in about 15% of recombinant CD2 molecules. After denaturation and refolding in the absence of the fusion partner, dimeric CD2 is converted to monomer, illustrating that the dimeric form represents a metastable folded state. The crystal structure of this dimeric form, refined to 2.O-A resolution, reveals two domains with overall similarity to the IgSF fold found in the monomer. However, in the dimer each domain is formed by the intercalation of two polypeptide chains. Hence each domain represents a distinct folding unit that can assemble in two different ways. In the dimer the two domains fold around a hydrophilic interface believed to mimic the cell adhesion interaction at the cell surface, and the formation of dimer can be regulated by mutating single residues at this interface. This unusual misfolded form of the protein, which appears to result from inter-rather than intramolecular interactions being favored by an intermediate structure formed during the folding process, illustrates that evolution of protein oligomers is possible from the sequence for a single protein domain.CD2 is a membrane glycoprotein present on the surface of T lymphocytes and is known to function as an adhesion molecule to promote T-cell activation (reviewed in ref. 1). The extracellular region contains two immunoglobulin superfamily (IgSF) domains for which the structure has been determined for rat and human CD2 by x-ray crystallography (2, 3) and for the NH2-terminal domain (CD2 D1) by NMR (4,5). The major ligands for CD2 are believed to have a similar structure with two IgSF domains and are CD48 in rodents (6, 7) and CD58 in humans (8, 9). Extensive mutagenesis studies indicate adhesion results from the association of the predominantly charged GFCC'C" faces of the NH2-terminal domains of CD2 and its ligands (10-13). Each of these domains lacks the intersheet disulfide bond found in most IgSF structures. An interaction believed to mimic this binding has been observed between adjacent molecules in the crystal lattices of both rat and human CD2 (2, 3). At the cell surface these interactions are of low affinity and are characterized by a particularly fast dissociation rate (14,15).The NMR structure of domain 1 of rat CD2 was determined from recombinant monomeric material prepared by expression of the domain as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. We have found that after cleavage of this domain from the GST there is a small amount of dimeric CD2 Dl, and we have determined its structure by x-ray crystallography.l This metastable dimeric form readily converts to monomer after unfolding, and the pro...