Mohammad Mubinur Rahman

Collagen prolyl 4-hydroxylase (C-P4H) is an essential enzyme in collagen synthesis and known to be a potential target for drugs that prevent excess collagen formation. Currently known C-P4H inhibitors target the catalytic site of C-P4H, being analogues of 2-oxoglutarate (2OG). However, in mammalian cells there are many other 2OG-dependent dioxygenases with a highly similar catalytic domain, which limits the selective specificity of drugs targeting C-P4H activity. The peptide-substrate-binding (PSB) domain is unique for the C-P4H family and known to be important for the catalytic efficiency of C-P4H. Therefore, interfering with peptide binding to the PSB domain might allow more specific inhibition of the hydroxylation activity of C-P4Hs. We developed a robust FRET-based high-throughput screening assay (Z’ > 0.73) based on PSB-peptide interactions. This assay was used to screen a peptidomimetic library of 15614 compounds with the PSB domains of C-P4H isoforms I and II. A hit compound (OUL-PSBi-001) was identified with IC ₅₀ s of 40 µM and 62 µM for PSB-I and PSB-II, respectively. We also showed that this compound indeed inhibits the catalytic activity of the full-length CP4H-I and -II. This FRET assay provides a new strategy for finding selective inhibitors for the treatment of fibrotic diseases and cancer.

Collagen prolyl 4‐hydroxylase (C‐P4H) catalyzes the 4‐hydroxylation of Y‐prolines of the XYG‐repeat of procollagen. C‐P4Hs are tetrameric α₂β₂ enzymes. The α‐subunit provides the N‐terminal dimerization domain, the middle peptide‐substrate–binding (PSB) domain, and the C‐terminal catalytic (CAT) domain. There are three isoforms of the α‐subunit, complexed with a β‐subunit that is protein disulfide isomerase, forming C‐P4H I‐III. The PSB domain of the α‐subunit binds proline‐rich peptides, but its function with respect to the prolyl hydroxylation mechanism is unknown. An extended mode of binding of proline‐rich peptides (PPII, polyproline type‐II, conformation) to the PSB‐I domain has previously been reported for the PPG‐PPG‐PPG and P9 peptides. Crystal structures now show that peptides with the motif PxGP (PPG‐PRG‐PPG, PPG‐PAG‐PPG) (where x, at Y‐position 5, is not a proline) bind to the PSB‐I domain differently, more deeply, in the peptide‐binding groove. The latter mode of binding has previously been reported for structures of the PSB‐II domain complexed with these PxGP‐peptides. In addition, it is shown here by crystallographic binding studies that the POG‐PAG‐POG peptide (with 4‐hydroxyprolines at Y‐positions 2 and 8) also adopts the PxGP mode of binding to PSB‐I as well as to PSB‐II. Calorimetric binding studies show that the affinities of these peptides are lower for PSB‐I than for PSB‐II, with, respectively, K_D values of about 70 μM for PSB‐I and 20 μM for PSB‐II. The importance of these results for understanding the reaction mechanism of C‐P4H, in particular concerning the function of the PSB domain, is discussed.

The Ilv/ED dehydratase protein family includes dihydroxy acid-, gluconate-, 6-phosphogluconate- and pentonate dehydratases. The members of this family are involved in various biosynthetic and carbohydrate metabolic pathways. Here, we describe the first crystal structure of D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) at 2.7 Å resolution and compare it with other available enzyme structures from the IlvD/EDD protein family. The quaternary structure of CcXyDHT is a tetramer, and each monomer is composed of two domains in which the N-terminal domain forms a binding site for a [2Fe-2S] cluster and a Mg²⁺ ion. The active site is located at the monomer-monomer interface and contains residues from both the N-terminal recognition helix and the C-terminus of the dimeric counterpart. The active site also contains a conserved Ser490, which probably acts as a base in catalysis. Importantly, the cysteines that participate in the binding and formation of the [2Fe-2S] cluster are not all conserved within the Ilv/ED dehydratase family, which suggests that some members of the IlvD/EDD family may bind different types of [Fe-S] clusters.

We present a novel crystal structure of the IlvD/EDD family enzyme, l-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT, EC 4.2.1.25), which catalyzes the conversion of l-arabinonate to 2-dehydro-3-deoxy-l-arabinonate. The enzyme is a tetramer consisting of a dimer of dimers, where each monomer is composed of two domains. The active site contains a catalytically important [2Fe-2S] cluster and Mg²⁺ ion and is buried between two domains, and also at the dimer interface. The active site Lys129 was found to be carbamylated. Ser480 and Thr482 were shown to be essential residues for catalysis, and the S480A mutant structure showed an unexpected open conformation in which the active site was more accessible for the substrate. This structure showed the partial binding of l-arabinonate, which allowed us to suggest that the alkoxide ion form of the Ser480 side chain functions as a base and the [2Fe-2S] cluster functions as a Lewis acid in the elimination reaction.

Summary Three pathogenic species of the genus Yersinia assemble adhesive fimbriae via the FGL-chaperone/usher pathway. Closely related Y. pestis and Y. pseudotuberculosis elaborate the pH6 antigen (Psa), which mediates bacterial attachment to alveolar cells of the lung. Y. enterocolitica, instead, assembles the homologous fimbriae Myf of unknown function. Here, we discovered that Myf, like Psa, specifically recognizes β1-3– or β1-4–linked galactose in glycosphingolipids, but completely lacks affinity for phosphatidylcholine, the main receptor for Psa in alveolar cells. The crystal structure of a subunit of Psa (PsaA) complexed with choline together with mutagenesis experiments revealed that PsaA has four phosphatidylcholine binding pockets that enable super-high-avidity binding of Psa-fibers to cell membranes. The pockets are arranged as six tyrosine residues, which are all missing in the MyfA subunit of Myf. On the other hand, the crystal structure of the MyfA-galactose complex revealed that the galactose-binding site is more extended in MyfA, enabling tighter binding to lactosyl moieties. Our results suggest that during evolution, Psa has acquired a tyrosine-rich surface that enables it to bind to phosphatidylcholine and mediate adhesion of Y. pestis/pseudotuberculosis to alveolar cells, whereas Myf has specialized as a carbohydrate-binding adhesin, facilitating the attachment of Y. enterocolitica to intestinal cells.

L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase fromRhizobium leguminosarumbv.trifolii(RlArDHT) and D-xylonate dehydratase fromCaulobacter crescentus(CcXyDHT) were heterologously expressed inEscherichia coliand purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals ofRlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space groupP2₁, with unit-cell parametersa = 106.07,b= 208.61,c= 147.09 Å, β = 90.43°. EightRlArDHT molecules (two tetramers) in the asymmetric unit give aV_Mvalue of 3.2 ų Da⁻¹and a solvent content of 62%. Crystals ofCcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space groupC2, with unit-cell parametersa= 270.42,b= 236.13,c = 65.17 Å, β = 97.38°. FourCcXyDHT molecules (a tetramer) in the asymmetric unit give aV_Mvalue of 4.0 ų Da⁻¹and a solvent content of 69%.

Summary: Coli surface antigen 6 (CS6) is a widely expressed enterotoxigenic Escherichia coli (ETEC) colonization factor that mediates bacterial attachment to the small intestinal epithelium. CS6 is a polymer of two protein subunits CssA and CssB, which are secreted and assembled on the cell surface via the CssC/CssD chaperone usher (CU) pathway. Here, we present an atomic resolution model for the structure of CS6 based on the results of X‐ray crystallographic, spectroscopic and biochemical studies, and suggest a mechanism for CS6‐mediated adhesion. We show that the CssA and CssB subunits are assembled alternately in linear fibres by the principle of donor strand complementation. This type of fibre assembly is novel for CU assembled adhesins. We also show that both subunits in the fibre bind to receptors on epithelial cells, and that CssB, but not CssA, specifically recognizes the extracellular matrix protein fibronectin. Taken together, structural and functional results suggest that CS6 is an adhesive organelle of a novel type, a hetero‐polyadhesin that is capable of polyvalent attachment to different receptors.