Dwight Moore

Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, α1β1 integrin and Staphylococcus aureus Cna MSCRAMM

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin α2β1 and a Staphylococcus aureus adhesin reveal a “trench” on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512–28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833–838). We report here the crystal structure of the α subunit I domain from the α1β1 integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human α1 integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.

A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor.

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.

Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D

The crystal structure of profactor D, determined at 2.1 Å resolution with an Rfree and an R‐factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N‐22, 71–76, 143–152, 187–193 and 215–223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen–active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin‐2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self‐inhibited active site conformation. Examination of the surface properties of the N‐terminus‐binding pocket indicates that Ile16 may play the initial positioning role for the N‐terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen‐like zymogens, is followed by a factor D‐unique step, the re‐orientation of an external Arg218 to an internal position for salt‐bridging with Asp189, leading to the generation of the self‐inhibited factor D.

New structural motifs on the chymotrypsin fold and their potential roles in complement factor B

Factor B and C2 are two central enzymes for complement activation. They are multidomain serine proteases and require cofactor binding for full expression of proteolytic activities. We present a 2.1 Å crystal structure of the serine protease domain of factor B. It shows a number of structural motifs novel to the chymotrypsin fold, which by sequence homology are probably present in C2 as well. These motifs distribute characteristically on the protein surface. Six loops surround the active site, four of which shape substrate‐binding pockets. Three loops next to the oxyanion hole, which typically mediate zymogen activation, are much shorter or absent. Three insertions including the linker to the preceding domain bulge from the side opposite to the active site. The catalytic triad and non‐specific substrate‐binding site display active conformations, but the oxyanion hole displays a zymogen‐like conformation. The bottom of the S1 pocket has a negative charge at residue 226 instead of the typical 189 position. These unique structural features may play different roles in domain–domain interaction, cofactor binding and substrate binding.

Crystallization and preliminary X-ray crystallographic analysis of Ace: a Collagen-binding MSCRAMM from Enterococcus faecalis

Ace is a collagen-binding bacterial cell surface adhesin from Enterococcus faecalis. The collagen-binding domain of Ace (termed Ace40) and its truncated form Ace19 have been crystallized by the vapor-diffusion hanging-drop method. Ace19 was crystallized in two different crystal forms. A complete 1.65 A data set has been collected on the orthorhombic crystal form with unit cell parameters a=38.43 b=48.91 and c=83.73 A. Ace40 was crystallized in the trigonal space group P3(1)21 or P3(2)21 with unit cell parameters a=b=80.24, c=105.91 A; alpha=beta=90 and gamma=120 degrees. A full set of X-ray diffraction data was collected to 2.5 A. Three heavy atom derivative data sets have been successfully obtained for Ace19 crystals and structural analysis is in progress.

Glucosamine 6‐phosphate deaminase in normal human erythrocytes

Summary In the course of an investigation of hexosamine catabolism in the human malaria parasite, Plasmodium falciparum, it became apparent that a basic understanding of the relevant enzymatic reactions in the host erythrocyte is lacking. To acquire the necessary basic knowledge, we have determined the activities of several enzymes involved in hexosamine metabolism in normal human red blood cells. In the present communication we report the results of studies of glucosamine 6‐phosphate deaminase (GlcN6‐P) using a newly developed sensitive radiometric assay. The mean specific activity in extracts of fresh erythrocytes assayed within 4h of collection was 14–7nmol/h/mg protein, whereas preparations from older erythrocytes that had been stored at 4°C for up to 4 weeks had a mean specific activity of 6–2 nmol/h/mg. Characterization of the deaminase by chromatofocusing gave a pi of 8–55. The enzyme was optimally active at pH 9–0 and had a Km of 41 MUM. The metal chelators EDTA and EGTA were non‐inhibitory; however, inhibition was observed in the presence of metal ions, especially Cu2+, Ni2+ and Zn2+. In addition, the deaminase was also inhibited by several sugar phosphates including the reaction product, fructose 6‐phosphate.

N-acetylglucosamine kinase and N-acetylglucosamine 6-phosphate deacetylase in normal human erythrocytes and Plasmodium falciparum.

The major pathways of glucose metabolism in the malaria parasite, Plasmodium falciparum, have now been elucidated, and the structures and properties of parasite‐specific enzymes are presently being investigated. Little is known, however, about the enzymes catalysing monosaccharide interconversions in the parasite. In the present investigation we have examined the pathway of N‐acetylglucosamine catabolism which, in higher organisms, involves the following reaction sequence: N‐acetylglucosamine → N‐acetylglucosamine 6‐phosphate → glucosamine 6‐phosphate →  fructose 6‐phosphate. Assay of the specific kinase (E.C. 2.7.1.59) catalysing the phosphorylation of the sugar showed that the enzyme is present in Plasmodium extracts as well as in normal human erythrocytes; specific activities of 7.2 and 5.3 nmol/h/mg protein were measured for the parasite and erythrocyte extracts, respectively. n‐Acetylglucosamine 6‐phosphate deacetylase (E.C. 3.5.1.25), catalysing the second reaction, was also detected in both normal and Plasmodium‐infected erythrocytes. At 75% parasitaemia, the deacetylase activity was close to 3 times higher than that of normal control cells. The erythrocyte deacetylase was purified approximately 16 000‐fold by chromatography on DE52 cellulose, chromatofocusing, and size exclusion chromatography. Attempts to purify the parasite enzyme by the same procedures were unsuccessful due to loss of activity. A partially purified erythrocyte deacetylase preparation (eluted from DE52 cellulose) had a pH optimum of 7.5, a pI of 6.0, as indicated by chromatofocusing, and a Km of 29 μM. In conjunction with previous investigations, the present study indicated that all three enzymes required for N‐acetylglucosamine utilization are present in Plasmodium parasites as well as in normal erythrocytes.

Crystallization and preliminary crystallographic investigation of porcine quinolinate phosphoribosyltransferase

Quinolinate phosphoribosyltransferase (QPRT), purified from hog liver, has been crystallized using PEG 8000 as the precipitant. The crystals form long hexagonal rods in the space group P6322 with cell dimensions a = b = 121.7, c = 94.5 A. Based on the unit-cell dimensions and the calculated molecular mass of 33 500 Da, the Matthews coefficient suggests one molecule per asymmetric unit (Vm = 3.45 A3 Da-1; 64% solvent). Three native data sets were collected to a resolution of 2.5 A and merged to provide a set that is 94.7% complete, with an Rsym value of 9.6%.