Andrew A. McCarthy

Structure of C3b reveals conformational changes that underlie complement activity.

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-Å resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 Å away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the α-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli.

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 × 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 Å resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.

X‐ray structure of human acid‐β‐glucosidase, the defective enzyme in Gaucher disease

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid‐β‐glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ∼200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non‐ neuronopathic and neuronopathic disease, respectively. We report the X‐ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α)8 TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α‐helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin‐like domain on which L444 is located, suggesting an important regulatory or structural role for this non‐catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme‐replacement therapy, and for designing structure‐based drugs aimed at restoring the activity of defective GlcCerase.

Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase.

Cysteinyl leukotrienes are key mediators in inflammation and have an important role in acute and chronic inflammatory diseases of the cardiovascular and respiratory systems, in particular bronchial asthma. In the biosynthesis of cysteinyl leukotrienes, conversion of arachidonic acid forms the unstable epoxide leukotriene A4 (LTA4). This intermediate is conjugated with glutathione (GSH) to produce leukotriene C4 (LTC4) in a reaction catalysed by LTC4 synthase: this reaction is the key step in cysteinyl leukotriene formation. Here we present the crystal structure of the human LTC4 synthase in its apo and GSH-complexed forms to 2.00 and 2.15 Å resolution, respectively. The structure reveals a homotrimer, where each monomer is composed of four transmembrane segments. The structure of the enzyme in complex with substrate reveals that the active site enforces a horseshoe-shaped conformation on GSH, and effectively positions the thiol group for activation by a nearby arginine at the membrane–enzyme interface. In addition, the structure provides a model for how the ω-end of the lipophilic co-substrate is pinned at one end of a hydrophobic cleft, providing a molecular ‘ruler’ to align the reactive epoxide at the thiol of glutathione. This provides new structural insights into the mechanism of LTC4 formation, and also suggests that the observed binding and activation of GSH might be common for a family of homologous proteins important for inflammatory and detoxification responses.

Structural Basis of HIV-1 Tethering to Membranes by the BST-2/Tetherin Ectodomain

The restriction factor BST-2/tetherin contains two membrane anchors employed to retain some enveloped viruses, including HIV-1 tethered to the plasma membrane in the absence of virus-encoded antagonists. The 2.77 A crystal structure of the BST-2/tetherin extracellular core presented here reveals a parallel 90 A long disulfide-linked coiled-coil domain, while the complete extracellular domain forms an extended 170 A long rod-like structure based on small-angle X-ray scattering data. Mutagenesis analyses indicate that both the coiled coil and the N-terminal region are required for retention of HIV-1, suggesting that the elongated structure can function as a molecular ruler to bridge long distances. The structure reveals substantial irregularities and instabilities throughout the coiled coil, which contribute to its low stability in the absence of disulfide bonds. We propose that the irregular coiled coil provides conformational flexibility, ensuring that BST-2/tetherin anchoring both in the plasma membrane and in the newly formed virus membrane is maintained during virus budding.

Structural Insights Into the Slit-Robo Complex.

Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1–2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.

MxCuBE: a synchrotron beamline control environment customized for macromolecular crystallography experiments

MxCuBE is a beamline control environment optimized for the needs of macromolecular crystallography. This paper describes the design of the software and the features that MxCuBE currently provides.

Structure and Function of the C-terminal Domain of the Polymerase Cofactor of Rabies Virus

The phosphoprotein (P) of rabies virus binds the viral polymerase to the nucleoprotein (N)-RNA template for transcription and replication. By limited protease digestion we defined a monomeric C-terminal domain of P that can bind to N-RNA. The atomic structure of this domain was determined and previously described mutations that interfere with binding of P to N-RNA could now be interpreted. There appears to be two features involved in this activity situated at opposite surfaces of the molecule: a positively charged patch and a hydrophobic pocket with an exposed tryptophan side-chain. Other previously published work suggests a conformational change in P when it binds to N-RNA, which may imply the repositioning of two helices that would expose a hydrophobic groove for interaction with N. This domain of rabies virus P is structurally unrelated to the N-RNA binding domains of the phosphoproteins of Sendai and measles virus that are members of the same order of viruses, the non-segmented negative strand RNA viruses. The implications of this finding for the evolution of this virus group are discussed.

Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold.

BACKGROUND Methylmalonyl-CoA epimerase (MMCE) is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Present in many bacteria and in animals, it catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B12-dependent enzyme, methylmalonyl-CoA mutase. Defects in this pathway can result in severe acidosis and cause damage to the central nervous system in humans. RESULTS The crystal structure of MMCE from Propionibacterium shermanii has been determined at 2.0 A resolution. The MMCE monomer is folded into two tandem betaalphabetabetabeta modules that pack edge-to-edge to generate an 8-stranded beta sheet. Two monomers then pack back-to-back to create a tightly associated dimer. In each monomer, the beta sheet curves around to create a deep cleft, in the floor of which His12, Gln65, His91, and Glu141 provide a binding site for a divalent metal ion, as shown by the binding of Co2+. Modeling 2-methylmalonate into the active site identifies two glutamate residues as the likely essential bases for the epimerization reaction. CONCLUSIONS The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.

A decade of user operation on the macromolecular crystallography MAD beamline ID14-4 at the ESRF

The improvement of the X-ray beam quality achieved on ID14-4 by the installation of new X-ray optical elements is described.