M. Kristian Koski

Mutational spectrum of D-bifunctional protein deficiency and structure- based genotype-phenotype analysis

D-bifunctional protein (DBP) deficiency is an autosomal recessive inborn error of peroxisomal fatty acid oxidation. The clinical presentation of DBP deficiency is usually very severe, but a few patients with a relatively mild presentation have been identified. In this article, we report the mutational spectrum of DBP deficiency on the basis of molecular analysis in 110 patients. We identified 61 different mutations by DBP cDNA analysis, 48 of which have not been reported previously. The predicted effects of the different disease-causing amino acid changes on protein structure were determined using the crystal structures of the (3R)-hydroxyacyl-coenzyme A (CoA) dehydrogenase unit of rat DBP and the 2-enoyl-CoA hydratase 2 unit and liganded sterol carrier protein 2-like unit of human DBP. The effects ranged from the replacement of catalytic amino acid residues or residues in direct contact with the substrate or cofactor to disturbances of protein folding or dimerization of the subunits. To study whether there is a genotype-phenotype correlation for DBP deficiency, these structure-based analyses were combined with extensive biochemical analyses of patient material (cultured skin fibroblasts and plasma) and available clinical information on the patients. We found that the effect of the mutations identified in patients with a relatively mild clinical and biochemical presentation was less detrimental to the protein structure than the effect of mutations identified in those with a very severe presentation. These results suggest that the amount of residual DBP activity correlates with the severity of the phenotype. From our data, we conclude that, on the basis of the predicted effect of the mutations on protein structure, a genotype-phenotype correlation exists for DBP deficiency.

The active site of an algal prolyl 4-hydroxylase has a large structural plasticity

Prolyl 4-hydroxylases (P4Hs) are 2-oxoglutarate dioxygenases that catalyze the hydroxylation of peptidyl prolines. They play an important role in collagen synthesis, oxygen homeostasis, and plant cell wall formation. We describe four structures of a P4H from the green alga Chlamydomonas reinhardtii, two of the apoenzyme at 1.93 and 2.90Å resolution, one complexed with the competitive inhibitor Zn2+, and one with Zn2+ and pyridine 2,4-dicarboxylate (which is an analogue of 2-oxoglutarate) at 1.85Å resolution. The structures reveal the double-stranded β-helix core fold (jellyroll motif), typical for 2-oxoglutarate dioxygenases. The catalytic site is at the center of an extended shallow groove lined by two flexible loops. Mutagenesis studies together with the crystallographic data indicate that this groove participates in the binding of the proline-rich peptide-substrates. It is discussed that the algal P4H and the catalytic domain of collagen P4Hs have notable structural similarities, suggesting that these enzymes form a separate structural subgroup of P4Hs different from the hypoxia-inducible factor P4Hs. Key structural differences between these two subgroups are described. These studies provide first insight into the structure-function relationships of the collagen P4Hs, which unlike the hypoxia-inducible factor P4Hs use proline-rich peptides as their substrates.

The Crystal Structure of an Algal Prolyl 4-Hydroxylase Complexed with a Proline-rich Peptide Reveals a Novel Buried Tripeptide Binding Motif

Plant and algal prolyl 4-hydroxylases (P4Hs) are key enzymes in the synthesis of cell wall components. These monomeric enzymes belong to the 2-oxoglutarate dependent superfamily of enzymes characterized by a conserved jelly-roll framework. This algal P4H has high sequence similarity to the catalytic domain of the vertebrate, tetrameric collagen P4Hs, whereas there are distinct sequence differences with the oxygen-sensing hypoxia-inducible factor P4H subfamily of enzymes. We present here a 1.98-Å crystal structure of the algal Chlamydomonas reinhardtii P4H-1 complexed with Zn2+ and a proline-rich (Ser-Pro)5 substrate. This ternary complex captures the competent mode of binding of the peptide substrate, being bound in a left-handed (poly)l-proline type II conformation in a tunnel shaped by two loops. These two loops are mostly disordered in the absence of the substrate. The importance of these loops for the function is confirmed by extensive mutagenesis, followed up by enzyme kinetic characterizations. These loops cover the central Ser-Pro-Ser tripeptide of the substrate such that the hydroxylation occurs in a highly buried space. This novel mode of binding does not depend on stacking interactions of the proline side chains with aromatic residues. Major conformational changes of the two peptide binding loops are predicted to be a key feature of the catalytic cycle. These conformational changes are probably triggered by the conformational switch of Tyr140, as induced by the hydroxylation of the proline residue. The importance of these findings for understanding the specific binding and hydroxylation of (X-Pro-Gly)n sequences by collagen P4Hs is also discussed.

Binary Structure of the Two-Domain (3R)-Hydroxyacyl-CoA Dehydrogenase from Rat Peroxisomal Multifunctional Enzyme Type 2 at 2.38 Å Resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.

The Structural Motifs for Substrate Binding and Dimerization of the α Subunit of Collagen Prolyl 4-Hydroxylase

Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the proline hydroxylation of procollagen, an essential modification in the maturation of collagens. C-P4H consists of two catalytic α subunits and two protein disulfide isomerase β subunits. The assembly of these subunits is unknown. The α subunit contains an N domain (1-143), a peptide-substrate-binding-domain (PSB, 144-244) and a catalytic domain (245-517). Here, we report the dimeric structure of the N-terminal region (1-244) of the α subunit. It is shown that the N domain has an important role in the assembly of the C-P4H tetramer, by forming an extended four-helix bundle that includes an antiparallel coiled-coil dimerization motif between the two α subunits. Complexes of this construct with a C-P4H inhibitor and substrate show the mode of peptide-binding to the PSB domain. Both peptides adopt a poly-(L)-proline-type-II helix conformation and bind in a curved, asymmetric groove lined by conserved tyrosines and an Arg-Asp salt bridge.

Human Δ3,Δ2‐enoyl‐CoA isomerase, type 2: a structural enzymology study on the catalytic role of its ACBP domain and helix‐10

The catalytic domain of the trimeric human Δ3,Δ2‐enoyl‐CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E‐ or 3Z‐enoyl‐CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E‐enoyl‐CoA, which is a key intermediate in the β‐oxidation pathway. The active site is covered by the C‐terminal helix‐10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl‐CoA binding protein (ACBP) domain. Small angle X‐ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X‐ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop‐2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl‐CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl‐CoA isomerase activity; however, the full‐length isomerase is more efficient. Structural enzymological studies of helix‐10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop‐2 and loop‐4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis.

Crystallization and preliminary crystallographic data of 2-enoyl-CoA hydratase 2 domain of Candida tropicalis peroxisomal multifunctional enzyme type 2

In yeast, the second and the third reaction of the fatty-acid beta-oxidation spiral are catalysed by peroxisomal multifunctional enzyme type 2 (Mfe2p/Fox2p). This protein has two (3R)-hydroxyacyl-CoA dehydrogenase domains and a C-terminal 2-enoyl-CoA hydratase 2 domain. Here, the purification, crystallization and X-ray diffraction analysis of the hydratase 2 domain [CtMfe2p(dh(a+b)Delta)] from Candida tropicalis Mfe2p is reported. CtMfe2p(dh(a+b)Delta) was overexpressed as an enzymatically active recombinant protein and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 178.57, b = 60.46, c = 130.85 A, beta = 94.48 degrees. Selenomethionine-labelled protein was used for a multi-wavelength anomalous dispersion (MAD) experiment. A three-wavelength data set suitable for MAD phasing was collected to 2.25 A resolution using synchrotron radiation.

Structural enzymology binding studies of the peptide-substrate-binding domain of human collagen prolyl 4-hydroxylase (type-II): High affinity peptides have a PxGP sequence motif: Crystal structures of the PSB domain of human C-P4H-II

The peptide‐substrate‐binding (PSB) domain of collagen prolyl 4‐hydroxylase (C‐P4H, an α2β2 tetramer) binds proline‐rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C‐P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C‐terminal domain of the α subunit). The crystal structures of the PSB domain of the human C‐P4H isoform II (PSB‐II) complexed with and without various short proline‐rich peptides are described. The comparison with the previously determined PSB‐I peptide complex structures shows that the C‐P4H‐I substrate peptide (PPG)3, has at most very weak affinity for PSB‐II, although it binds with high affinity to PSB‐I. The replacement of the middle PPG triplet of (PPG)3 to the nonhydroxylatable PAG, PRG, or PEG triplet, increases greatly the affinity of PSB‐II for these peptides, leading to a deeper mode of binding, as compared to the previously determined PSB‐I peptide complexes. In these PSB‐II complexes, the two peptidyl prolines of its central P(A/R/E)GP region bind in the Pro5 and Pro8 binding pockets of the PSB peptide‐binding groove, and direct hydrogen bonds are formed between the peptide and the side chains of the highly conserved residues Tyr158, Arg223, and Asn227, replacing water mediated interactions in the corresponding PSB‐I complex. These results suggest that PxGP (where x is not a proline) is the common motif of proline‐rich peptide sequences that bind with high affinity to PSB‐II.

The extended structure of the periplasmic region of CdsD, a structural protein of the type III secretion system of Chlamydia trachomatis.

The type III secretion system (T3SS) is required for the virulence of many gram‐negative bacterial human pathogens. It is composed of several structural proteins, forming the secretion needle and its basis, the basal body. In Chlamydia spp., the T3SS inner membrane ring (IM‐ring) of the basal body is formed by the periplasmic part of CdsD (outer ring) and CdsJ (inner ring). Here we describe the crystal structure of the C‐terminal, periplasmic part of CdsD, not including the last 60 residues. Two crystal forms were obtained, grown in three different crystallization conditions. In both crystal forms there is one molecule per asymmetric unit adopting a similar extended structure. The structures consist of three periplasmic domains (PDs) of similar αββαβ topology as seen also in the structures of the homologous PrgH (Salmonella typhimurium) and YscD (Yersinia enterocolitica). Only in the C2 crystal form, there is a C‐terminal additional helix after the PD3 domain. The relative orientation of the three subsequent CdsD PD domains with respect to each other is more extended than in PrgH but less extended than in YscD. Small‐angle X‐ray scattering data show that also in solution this CdsD construct adopts the same elongated shape. In both crystal forms the CdsD molecules are packed in a parallel fashion, using translational crystallographic symmetry. The most extensive crystal contacts are preserved in both crystal forms, suggesting a possible mode of assembly of the CdsD periplasmic part into a 24‐mer complex forming the outer ring of the IM‐ring of the T3SS.