Songlin Li

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase - Structures of complexes with the substrate

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniaehyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-Å resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn349, His399, and Tyr408, is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the α- and β-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.

Crystal Structure of the Cytoskeleton-associated Protein Glycine-rich (CAP-Gly) Domain*

Cytoskeleton-associated proteins (CAPs) are involved in the organization of microtubules and transportation of vesicles and organelles along the cytoskeletal network. A conserved motif, CAP-Gly, has been identified in a number of CAPs, including CLIP-170 and dynactins. The crystal structure of the CAP-Gly domain ofCaenorhabditis elegans F53F4.3 protein, solved by single wavelength sulfur-anomalous phasing, revealed a novel protein fold containing three β-sheets. The most conserved sequence, GKNDG, is located in two consecutive sharp turns on the surface, forming the entrance to a groove. Residues in the groove are highly conserved as measured from the information content of the aligned sequences. The C-terminal tail of another molecule in the crystal is bound in this groove.

Structural genomics of Caenorhabditis elegans: crystal structure of the tropomodulin C-terminal domain

Shanyun Lu, Jindrich Symersky, Songlin Li, Mike Carson, Liqing Chen, Edward Meehan, and Ming Luo* Southeast Collaboratory for Structural Genomics, Center for Biophysical Science and Egineering, University of Alabama at Birmingham, Birmingham, Alabama Department of Chemistry, University of Alabama at Huntsville, Huntsville, Alabama Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama

Structural genomics of caenorhabditis elegans: Crystal structure of calmodulin

Introduction. Calmodulin (CaM), a conserved eucaryotic protein, can bind specifically to a large number of intracellular proteins and modulate their activity in response to the Ca concentration. This small 17-kDa acidic protein belongs to a family of homologous calcium-binding proteins that bind Ca through the EF-hand motif (e.g., parvalbumin or troponin C). A compact, calcium-free, apo form of CaM is converted to an extended dumbbellshaped form on binding Ca . The extended conformation of CaM has been by far the most thoroughly studied, especially by X-ray crystallography. It consists of two structurally similar domains separated by a flexible 28-residue helix. Each domain has two EF-hand motifs with bound Ca . The calciuminduced extension of CaM exposes two hydrophobic pockets, one per domain, which represent the binding sites for target proteins. In some protein targets, the CaM-binding region was located to a sequence of 18 amino acids predicted to form an -helix. On binding to the protein target, the central CaM helix unwinds, and the two hydrophobic pockets wrap around the -helix of the protein target. Structural plasticity of the hydrophobic pockets and flexibility of the central helix are thought to account for the ability of CaM to interact with a variety of different targets in a sequence-independent fashion. We have determined the crystal structure of calciumbound CaM from Caenorhabditis elegans (ceCaM) as a part of the Structural Genomics of C. elegans project. Besides the conserved features typical for all CaM’s, the ceCaM structure has the straightest central helix so far observed in CaM’s. This relatively straight helix may be induced by different crystallization conditions and/or by the crystal symmetry.

Structural genomics of Caenorhabditis elegans: Triosephosphate isomerase

Introduction. Triosephosphate isomerase (TIM, E.C. 5.3.1.1) catalyzes the reversible isomerization of dihydroxyacetone phosphate to D-glyceraldehyde 3-phosphate in the glycolytic pathway. It is a well-studied enzyme conserved in function across eukarya, bacteria, and archaea. The “TIM barrel” represents one of the most common protein folds and has been found in variety of proteins with different functions. All known TIM structures comprise approximately 250 amino acid residues per monomer and function as homodimers. It has been proposed that the isomerization reaction proceeds through an enediolate intermediate formed by substrate deprotonation. Crystal structures of TIM and its complexes with transition-state analogs have revealed conformational changes associated with substrate binding and a glutamate residue conveniently positioned to abstract and transfer a proton from one carbon of the substrate to another. The crystal structure of TIM from Caenorhabditis elegans (ceTIM) presented here was solved as a part of the structural genomics project on the C. elegans genome with 19,099 predicted genes. We employed a recently described phasing approach based on derivatization with halides, which seems to be applicable for high-throughput crystallographic projects. Both crystallographically independent ceTIM molecules have been found in the closed conformation with one sulfate and one acetate ion in the substratebinding site. The sidechain of the catalytic residue Glu164 has been refined in a dual conformation, which is relevant for the mechanism of proton transfer. Experimental. The protein expression in Escherichia coli and purification were performed as reported previously (also see http://www.sgce.cbse.uab.edu). As a result of Gateway cloning, the protein was produced with a hexahistidine tag and an eight-amino-acid peptide at both the N-terminus and the C-terminus. Initial crystallization conditions were obtained with the screening kit WIZARD I (Emerald BioStructures) and also with NATRIX (Hampton Research). Diffraction-grade crystals were grown at 22°C by vapor diffusion in hanging drops consisting of 3 L protein solution and 3 L well solution (2 M ammonium sulfate and 50 mM sodium acetate buffer, pH 5.5). The protein stock solution at 16 mg/mL was in 15 mM sodium N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), pH 7.5. The crystals are monoclinic, space group P21, a 36.40 Å, b 64.37 Å, c 105.71 Å, 91.5°, and the asymmetric unit contains two protein molecules. One such crystal was soaked for 10 min in a mother liquor with 0.5 M sodium iodide, dipped in mother liquor with 25% (v/v) glycerol, and flash-frozen in liquid nitrogen. Anomalous data to 2-Å resolution were collected at 170°C on an Raxis IV using the CuK radiation generated by a rotating anode (Table I). No attempts were made to collect the Bijvoet pairs close in time. The data were processed in Denzo/Scalepack, and the structure was solved by the single-wavelength anomolous diffraction (SAD) method in SOLVE. The anomalous Patterson map revealed 12 iodide sites with partial occupancies that were used for protein phasing. After density modification in RESOLVE, the refined phases provided a quality map suitable for automatic model building of approximately 75% of the amino acid residues by RESOLVE. A native diffraction data set was collected to 1.7 Å at the Stanford Synchrotron Radiation Laboratory (SSRL) beam line 9-1, with the wavelength at 0.976 Å. The native data were processed in HKL2000 and used for further structure refinement in Crystallography & NMR System (CNS). The final model [Protein Data Bank (PDB) code 1MO0] consists of two protein chains with a total of 502 amino acid residues, 461 water molecules, 5 sulfate ions, and 2 acetate ions. The crystallographic R-factor is 18.3%, and R-free is 21.3%, with no cutoffs. The model was validated in MolProbity and meets standards for the high-resolution structures. Results and Discussion. Soaking with iodide proved to be a quick and robust phasing method that can even use data collected on an in-house X-ray source. The refinement against native data resulted in defined protein chains without breaks in the 2Fo-Fc electron density map at 1.1 level. However, the Nand C-terminal peptides from the expression vector were just partially resolved in chain A

Structural genomics of Caenorhabditis elegans: structure of the BAG domain.

Binding of the BAG domain to the eukaryotic chaperone heat-shock protein (Hsp70) promotes ATP-dependent release of the protein substrate from Hsp70. Although the murine and human BAG domains have been shown to form an antiparallel three-helix bundle, the Caenorhabditis elegans BAG domain is formed by two antiparallel helices, while the third helix is extended away and stabilized by crystal-packing interactions. A small beta-sheet between helices 2 and 3 interferes with formation of the intramolecular three-helix bundle. However, intermolecular three-helix bundles are observed throughout the crystal packing and suggest that stable functional dimers and tetramers can be formed in solution. The structure may represent a new folding type of the BAG domain.

Structural Genomics of Caenorhabditis elegans: Structure of Dihydropteridine Reductase

Introduction. The biosynthesis of catecholamines includes hydroxylation of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Corresponding hydroxylases use tetrahydrobiopterin (BH4), which is oxidized to a quinonoid form of dihydrobiopterin (qBH2). Dihydropteridine reductase (DHPR) then uses NADH as a cofactor to reduce qBH2 back to BH4. The reaction consists of a protonation of the qBH2 substrate and a hydride transfer from NADH.

Purification, nanocrystallization and preliminary X-ray analysis of a C-terminal part of tropomodulin protein 1, isoform A, from Caenorhabditis elegans.

The C-terminal part of tropomodulin protein 1, isoform A, from Caenorhabditis elegans was expressed in Escherichia coli and purified to homogeneity. Optimized from the initial nanoscreen, crystals grew to dimensions of 0.25 x 0.15 x 0.15 mm at 277 K using 28.0%(v/v) PEG 400 as the precipitant by the hanging-drop vapor-diffusion technique. A data set of 94.9% completeness was collected to a resolution of 1.98 A at 100 K using a synchrotron X-ray source (SER-CAT). The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 31.7, b = 50.6, c = 107.1 A, and contained one molecule per asymmetric unit.

Expression, purification, crystallization of fragments from the C-terminal region of DFF45/ICAD.

DFF45/ICAD, which forms a heterodimer with DFF40/CAD as its DNase inhibitor and chaperone, plays a key role in nuclei DNA fragmentation in apoptosis. Several fragments from the C-terminal region of DFF45/ICAD have been cloned and expressed in Escherichia coli as His-tagged proteins. After purification to homogeneity, the recombinant proteins of three fragments were crystallized by the hanging-drop vapor-diffusion method. Of these, a crystal of DFF45c1 diffracted to 3.4 A in a capillary at 277 K and crystals of DFF45c2 diffracted to 3.2 A at cryotemperature using synchrotron radiation.