Jasbir Seehra

Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses.

Optimal immune responses require both an antigen-specific and a co-stimulatory signal. The shared ligands B7-1 and B7-2 on antigen-presenting cells deliver the co-stimulatory signal through CD28 and CTLA-4 on T cells. Signalling through CD28 augments the T-cell response, whereas CTLA-4 signalling attenuates it. Numerous animal studies and recent clinical trials indicate that manipulating these interactions holds considerable promise for immunotherapy. With the consequences of these signals well established, and details of the downstream signalling events emerging, understanding the molecular nature of these extracellular interactions becomes crucial. Here we report the crystal structure of the human CTLA-4/B7-1 co-stimulatory complex at 3.0 Å resolution. In contrast to other interacting cell-surface molecules, the relatively small CTLA-4/B7-1 binding interface exhibits an unusually high degree of shape complementarity. CTLA-4 forms homodimers through a newly defined interface of highly conserved residues. In the crystal lattice, CTLA-4 and B7-1 pack in a strikingly periodic arrangement in which bivalent CTLA-4 homodimers bridge bivalent B7-1 homodimers. This zipper-like oligomerization provides the structural basis for forming unusually stable signalling complexes at the T-cell surface, underscoring the importance of potent inhibitory signalling in human immune responses.

A Novel Cytosolic Calcium-independent Phospholipase A2 Contains Eight Ankyrin Motifs

We report the purification, molecular cloning, and expression of a novel cytosolic calcium-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells, which lacks extended homology to other phospholipases. iPLA2 is an 85-kDa protein that exists as a multimeric complex of 270-350 kDa with a specific activity of 1 μmol/min/mg. The full-length cDNA clone encodes a 752-amino acid cytoplasmic protein with one lipase motif (GXS465XG) and eight ankyrin repeats. Expression of the cDNA in mammalian cells generates an active 85-kDa protein. Mutagenesis studies show that Ser465 and the ankyrin repeats are required for activity. We demonstrate that iPLA2 selectively hydrolyzes the sn-2 over sn-1 fatty acid by 5-fold for 1,2-dipalmitoyl phosphatidylcholine in a mixed micelle. Moreover, we found the fatty acid preference at the sn-2 position to be highly dependent upon substrate presentation. However, iPLA2 does have a marked preference for 1,2-dipalmitoyl phosphatidic acid presented in a vesicle, generating the lipid second messenger lysophosphatidic acid. Finally the enzyme is able to hydrolyze the acetyl moiety at the sn-2 position of platelet-activating factor.

Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism.

Cytosolic phospholipase A2 initiates the biosynthesis of prostaglandins, leukotrienes, and platelet-activating factor (PAF), mediators of the pathophysiology of asthma and arthritis. Here, we report the X-ray crystal structure of human cPLA2 at 2.5 A. cPLA2 consists of an N-terminal calcium-dependent lipid-binding/C2 domain and a catalytic unit whose topology is distinct from that of other lipases. An unusual Ser-Asp dyad located in a deep cleft at the center of a predominantly hydrophobic funnel selectively cleaves arachidonyl phospholipids. The structure reveals a flexible lid that must move to allow substrate access to the active site, thus explaining the interfacial activation of this important lipase.

The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography.

In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane‐anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division. Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA‐FtsZ interaction is mediated by their C‐terminal domains. We present the X‐ray crystal structures of the C‐terminal FtsZ‐binding domain of ZipA and a complex between this domain and a C‐terminal fragment of FtsZ. The ZipA domain is a six‐stranded β‐sheet packed against three α‐helices and contains the split β‐α‐β motif found in many RNA‐binding proteins. The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent. In the complex, the 17‐residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended β‐strand followed by α‐helix. An alanine‐scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA.

1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling.

Interleukin 6 (IL‐6) has many biological activities in vivo, and deregulation has been implicated in many disease processes. IL‐6, a 185 amino acid polypeptide was refolded, purified and crystallized. The crystals diffracted to beyond 1.9 Å and the structure was solved using single isomorphous replacement. The X‐ray structure of IL‐6 is composed of a four helix bundle linked by loops and an additional mini‐helix. 157 out of 185 residues are well defined in the final structure, with 18 N‐terminal and 8 A–B loop amino acids displaying no interpretable electron density. The three‐dimensional structure has been used to construct a model of IL‐6 interacting with the IL‐6 receptor (α‐chain) and gp130 (β‐chain) that gives new insight into the process of molecular recognition and signaling. Based on this model, we predict a fourth binding site on IL‐6, a low affinity IL‐6–IL‐6 interaction, which may be necessary for the sequential assembly of a functional hexameric IL‐6 receptor complex.

Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.

Complete 1H, 15N and 13C assignments, secondary structure, and topology of recombinant human interleukin-6.

SummaryEssentially complete backbone and side-chain 1H, 15N and 13C resonance assignments for the 185-aminoacid cytokine interleukin-6 (IL-6) are presented. NMR experiments were performed on uniformly [15N]-and [15N, 13C]-labeled recombinant human IL-6 (rIL-6) using a variety of heteronuclear NMR experiments. A combination of 13C-chemical shift, amide hydrogen-bond exchange, and 15N-edited NOESY data allowed for analysis of the secondary structure of IL-6. The observed secondary structure of IL-6 is composed of loop regions connecting five α-helices, four of which are consistent in their length and disposition with the four-helix bundle motif present in other related cytokines and previously postulated for IL-6. In addition, the topology of the overall fold was found to be consistent with a left-handed up-up-down-down four-helix bundle based on a number of long-range interhelical NOEs. The results presented here provide deeper insight into structure-function relationships among members of the four-helix bundle family of proteins.

correction: Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses

This corrects the article DOI: 35069118