Karl Harlos

Structural basis for the recognition of hydroxyproline in HIF-1|[alpha]| by pVHL

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1α is the oxygen-regulated subunit of HIF-1, an αβ heterodimeric complex. HIF-1α is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel–Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1α by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1α. The crystal structure of a hydroxylated HIF-1α peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

A functional and structural basis for TCR cross-reactivity in multiple sclerosis

The multiple sclerosis (MS)-associated HLA major histocompatibility complex (MHC) class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show that together the DRB1 and DRB5 loci may influence susceptibility to MS. We demonstrate that a T cell receptor (TCR) from an MS patient recognized both a DRB1*1501-restricted myelin basic protein (MBP) and DRB5*0101-restricted Epstein-Barr virus (EBV) peptide. Crystal structure determination of the DRB5*0101-EBV peptide complex revealed a marked degree of structural equivalence to the DRB1*1501–MBP peptide complex at the surface presented for TCR recognition. This provides structural evidence for molecular mimicry involving HLA molecules. The structural details suggest an explanation for the preponderance of MHC class II associations in HLA-associated diseases.

Tick Histamine-Binding Proteins: Isolation, Cloning, and Three-Dimensional Structure

High-affinity histamine-binding proteins (HBPs) were discovered in the saliva of Rhipicephalus appendiculatus ticks. Their ability to outcompete histamine receptors indicates that they suppress inflammation during blood feeding. The crystal structure of a histamine-bound HBP, determined at 1.25 A resolution, reveals a lipocalin fold novel in containing two binding sites for the same ligand. The sites are orthogonally arranged and highly rigid and form an internal surface of unusual polar character that complements the physicochemical properties of histamine. As soluble receptors of histamine, HBPs offer a new strategy for controlling histamine-based diseases.

Structure of human CD1b with bound ligands at 2.3 Å, a maze for alkyl chains

The human genome encodes five nonpolymorphic major histocompatibility complex class I–like glycoproteins, CD1a to CD1e, that present lipid antigens for specific recognition by T lymphocytes. Using single alkyl chain detergents, we developed a protocol to generate recombinant human CD1b-lipid complexes. We present here the crystal structures of CD1b in complex with either phosphatidylinositol or ganglioside GM2 at 2.3 Å and 2.8 Å resolutions, respectively. The antigen-binding groove houses four interlinked hydrophobic channels that are occupied by the alkyl chains of the glycolipid plus two detergent molecules. A distinct exit beneath the α2 helix further contributes to the plasticity of the binding groove. These structures reveal the mechanism by which two alkyl chain lipids bind to CD1b, and how CD1b can adapt to ligands of different alkyl chain length. They also suggest how very long alkyl chains, such as those of mycolic acid, could be fully contained within the binding groove. These results extend the spectrum of potential CD1b ligands by revealing that, in addition to two alkyl chain lipids, mono-alkyl and triple-alkyl chain lipids can be accommodated in the binding groove.

Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase.

Clavaminate synthase (CAS), a remarkable Fe(II)/2-oxoglutarate oxygenase, catalyzes three separate oxidative reactions in the biosynthesis of clavulanic acid, a clinically used inhibitor of serine β-lactamases. The first CAS-catalyzed step (hydroxylation) is separated from the latter two (oxidative cyclization/desaturation) by the action of an amidinohydrolase. Here, we describe crystal structures of CAS in complex with Fe(II), 2-oxoglutarate (2OG) and substrates (N-α-acetyl-L-arginine and proclavaminic acid). They reveal how CAS catalyzes formation of the clavam nucleus, via a process unprecedented in synthetic organic chemistry, and suggest how it discriminates between substrates and controls reaction of its highly reactive ferryl intermediate. The presence of an unpredicted jelly roll β-barrel core in CAS implies divergent evolution within the family of 2OG and related oxygenases. Comparison with other non-heme oxidases/oxygenases reveals flexibility in the position which dioxygen ligates to the iron, in contrast to the analogous heme-using enzymes.

Structure and dimerization of a soluble form of B7-1.

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Control of the structure and fluidity of phosphatidylglycerol bilayers by pH titration

Abstract Titration of the single dissociable proton in phosphatidylglycerol bilayers not only shifts the ordered-fluid phase transition but also changes the bilayer fluidity in the region above the phase transition, and gives rise to a quite different bilayer structure in the region below the phase transition: 1. 1. The ordered-fluid phase transition temperatures of dimyristoyl and dipalmitoyl phosphatidylglycerol bilayers have been measured as a function of bulk pH in 0.1 M salt using water-lipid partitioning spin labels. From the dissociation curve obtained, it is found that the one titrable proton has an apparent p K a of 2.9 for bilayers of both lipids. 2. 2. In the fully ionized state, these phosphatidylglycerols not only give very similar transition temperatures (23°C for dimyristoyl and 40°C for dipalmitoyl chains) and pre-transition temperatures to the similar chain length phosphatidylcholines, but also show identical bilayer structures at corresponding temperatures when examined by freeze-fracture electron microscopy: i.e. defects, ripples and jumbled patterns, in the ordered, pre-transitional and fluid phases, respectively. 3. 3. When the phosphatidylglycerols are fully protonated, the bilayer pre-transition, as monitored by the spin labels, is absent and the main transition is somewhat broader and increased in temperature by approx. 17°C. Exclusively smooth bilayers are observed by electron microscopy at all temperatures. This suggests that, contrary to the situation in the charged bilayers, the phosphatidylglycerol molecules are not tilted relative to the bilayer normal in the ordered phase. 4. 4. In fluid bilayers, above the main transition temperature, the fluidity is found to be greater when the phosphatidylglycerol molecules are charged than when uncharged, due to the increased intermolecular separation caused by electrostatic repulsion. The results demonstrate that the structure and fluidity of charged lipid bilayer membranes can be changed isothermally without the mediation of the ordered-fluid phase transition.

A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high‐throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre‐scale sitting‐drop vapour‐diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96‐well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre‐programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information‐management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web‐based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre‐scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Structural Basis of Substrate Discrimination and Integrin Binding by Autotaxin.

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B–like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

Bound Water Structure and Polymorphic Amino Acids Act Together to Allow the Binding of Different Peptides to MHC Class I HLA-B53

The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes.