Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs. Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomonas species (PAP), solved to 2.1- and 1.96-A resolution, respectively. Comparative studies of PAP structures with mesophilic counterparts show that the overall structures are similar but that the conformation of the substrate-free active site in PAP resembles that of the substrate-bound region of the mesophilic homolog, with both an active-site tyrosine and a substrate-binding loop displaying a conformation as in the substrate-bound form of the mesophilic proteases. Further, a region in the catalytic domain of PAP undergoes a conformational change with a loop movement as large as 13 A, induced by the binding of an extra calcium ion. Finally, the active site is more accessible due to deletions occurring in surrounding loop regions.     

Crystal structures of saposins A and C

Saposins A and C are sphingolipid activator proteins required for the lysosomal breakdown of galactosylceramide and glucosylceramide, respectively. The saposins interact with lipids, leading to an enhanced accessibility of the lipid headgroups to their cognate hydrolases. We have determined the crystal structures of human saposins A and C to 2.0 Angstroms and 2.4 Angstroms, respectively, and both reveal the compact, monomeric saposin fold. We confirmed that these two proteins were monomeric in solution at pH 7.0 by analytical centrifugation. However, at pH 4.8, in the presence of the detergent C(8)E(5), saposin A assembled into dimers, while saposin C formed trimers. Saposin B was dimeric under all conditions tested. The self-association of the saposins is likely to be relevant to how these small proteins interact with lipids, membranes, and hydrolase enzymes.     

Functional clues from the crystal structure of an orphan periplasmic ligand‐binding protein from Treponema pallidum

The spirochete Treponema pallidum is the causative agent of syphilis, a sexually transmitted infection of major global importance. Other closely related subspecies of Treponema also are the etiological agents of the endemic treponematoses, such as yaws, pinta, and bejel. The inability of T. pallidum and its close relatives to be cultured in vitro has prompted efforts to characterize T. pallidum's proteins structurally and biophysically, particularly those potentially relevant to treponemal membrane biology, with the goal of possibly revealing the functions of those proteins. This report describes the structure of the treponemal protein Tp0737; this polypeptide has a fold characteristic of a class of periplasmic ligand‐binding proteins associated with ABC‐type transporters. Although no ligand for the protein was observed in electron‐density maps, and thus the nature of the native ligand remains obscure, the structural data described herein provide a foundation for further efforts to elucidate the ligand and thus the function of this protein in T. pallidum.     

Structural studies of codeinone reductase reveal novel insights into aldo-keto reductase function in benzylisoquinoline alkaloid biosynthesis

Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the β1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway.     

Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism

Creatininase from Pseudomonas putida is a member of the urease-related amidohydrolase superfamily. The crystal structure of the Mn-activated enzyme has been solved by the single isomorphous replacement method at 1.8A resolution. The structures of the native creatininase and the Mn-activated creatininase-creatine complex have been determined by a difference Fourier method at 1.85 A and 1.6 A resolution, respectively. We found the disc-shaped hexamer to be roughly 100 A in diameter and 50 A in thickness and arranged as a trimer of dimers with 32 (D3) point group symmetry. The enzyme is a typical Zn2+ enzyme with a binuclear metal center (metal1 and metal2). Atomic absorption spectrometry and X-ray crystallography revealed that Zn2+ at metal1 (Zn1) was easily replaced with Mn2+ (Mn1). In the case of the Mn-activated enzyme, metal1 (Mn1) has a square-pyramidal geometry bound to three protein ligands of Glu34, Asp45, and His120 and two water molecules. Metal2 (Zn2) has a well-ordered tetrahedral geometry bound to the three protein ligands of His36, Asp45, and Glu183 and a water molecule. The crystal structure of the Mn-activated creatininase-creatine complex, which is the first structure as the enzyme-substrate/inhibitor complex of creatininase, reveals that significant conformation changes occur at the flap (between the alpha5 helix and the alpha6 helix) of the active site and the creatine is accommodated in a hydrophobic pocket consisting of Trp174, Trp154, Tyr121, Phe182, Tyr153, and Gly119. The high-resolution crystal structure of the creatininase-creatine complex enables us to identify two water molecules (Wat1 and Wat2) that are possibly essential for the catalytic mechanism of the enzyme. The structure and proposed catalytic mechanism of the creatininase are different from those of urease-related amidohydrolase superfamily enzymes. We propose a new two-step catalytic mechanism possibly common to creatininases in which the Wat1 acts as the attacking nucleophile in the water-adding step and the Wat2 acts as the catalytic acid in the ring-opening step.     

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s⁰_20,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier R_G values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.     

Structural insight into equine lentivirus receptor 1

Equine lentivirus receptor 1 (ELR1) has been identified as a functional cellular receptor for equine infectious anemia virus (EIAV). Herein, recombinant ELR1 and EIAV surface glycoprotein gp90 were respectively expressed in Drosophila melanogaster S2 cells, and purified to homogeneity by Ni-NTA affinity chromatography and gel filtration chromatography. Gel filtration chromatography and analytical ultracentrifugation (AUC) analyses indicated that both ELR1 and gp90 existed as individual monomers in solution and formed a complex with a stoichiometry of 1:1 when mixed. The structure of ELR1 was first determined with the molecular replacement method, which belongs to the space group P4₂2₁2 with one molecule in an asymmetric unit. It contains eight antiparallel β-sheets, of which four are in cysteine rich domain 1 (CRD1) and two are in CRD2 and CRD3, respectively. Alignment of ELR1 with HVEM and CD134 indicated that Tyr61, Leu70, and Gly72 in CRD1 of ELR1 are important residues for binding to gp90. Isothermal titration calorimetry (ITC) experiments further confirmed that Leu70 and Gly72 are the critical residues.     

The solution structure of the complement deregulator FHR5 reveals a compact dimer and provides new insights into CFHR5 nephropathy

The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (R_g) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.     

Crystal structures of methionine adenosyltransferase complexed with substrates and products reveal the methionine-ATP recognition and give insights into the catalytic mechanism

Methionine adenosyltransferases (MATs) are a family of enzymes in charge of synthesising S-adenosylmethionine (SAM), the most important methyl donor present in living organisms. These enzymes use methionine and ATP as reaction substrates, which react in a S(N)2 fashion where the sulphur atom from methionine attacks C5' from ATP while triphosphate chain is cleaved. A MAT liver specific isoenzyme has been detected, which exists in two distinct oligomeric forms, a dimer (MAT III) and a tetramer (MAT I). Our previously reported crystal structure of MAT I complexed with an inhibitor led to the identification of the methionine-binding site. We present here the results obtained from the complex of MAT I with a competitive inhibitor of methionine, (2S,4S)-amino-4,5-epoxypentanoic acid (AEP), which presents the same features at the methionine binding site reported before. We have also analysed several complexes of this enzyme with methionine and ATP and analogues of them, in order to characterise the interaction that is produced between both substrates. The crystal structures of the complexes reveal how the substrates recognise each other at the active site of the enzyme, and suggest a putative binding site for the product SAM. The residues involved in the interactions of substrates and products with MAT have been identified, and the results agree with all the previous data concerning mutagenesis experiments and crystallographic work. Moreover, all the information provided from the analysis of the complexes has allowed us to postulate a catalytic mechanism for this family of enzymes. In particular, we propose a key role for Lys182 in the correct positioning of the substrates, and Asp135(*), in stabilising the sulphonium group formed in the product (SAM).     

Order within disorder: Aggrecan chondroitin sulphate‐attachment region provides new structural insights into protein sequences classified as disordered

Structural investigation of proteins containing large stretches of sequences without predicted secondary structure is the focus of much increased attention. Here, we have produced an unglycosylated 30 kDa peptide from the chondroitin sulphate (CS)‐attachment region of human aggrecan (CS‐peptide), which was predicted to be intrinsically disordered and compared its structure with the adjacent aggrecan G3 domain. Biophysical analyses, including analytical ultracentrifugation, light scattering, and circular dichroism showed that the CS‐peptide had an elongated and stiffened conformation in contrast to the globular G3 domain. The results suggested that it contained significant secondary structure, which was sensitive to urea, and we propose that the CS‐peptide forms an elongated wormlike molecule based on a dynamic range of energetically equivalent secondary structures stabilized by hydrogen bonds. The dimensions of the structure predicted from small‐angle X‐ray scattering analysis were compatible with EM images of fully glycosylated aggrecan and a partly glycosylated aggrecan CS2‐G3 construct. The semiordered structure identified in CS‐peptide was not predicted by common structural algorithms and identified a potentially distinct class of semiordered structure within sequences currently identified as disordered. Sequence comparisons suggested some evidence for comparable structures in proteins encoded by other genes (PRG4, MUC5B, and CBP). The function of these semiordered sequences may serve to spatially position attached folded modules and/or to present polypeptides for modification, such as glycosylation, and to provide templates for the multiple pleiotropic interactions proposed for disordered proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg²⁺‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl₂, but became disrupted in the absence of MgCl₂, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.     

High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: New insights into the active site and the proton transfer pathways

The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 Å resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a₃ iron and Cu_B of the active site is fitted best by a peroxo-group or a chloride ion. Two waters or OH⁻ groups do not fit, one water (or OH⁻) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu_B ligand His326 is present in the imidazolate form.     

Crystal structures of human NUDT5 reveal insights into the structural basis of the substrate specificity

Human NUDT5 (hNUDT5) is an ADP-ribose pyrophosphatase (ADPRase) belonging to the Nudix hydrolase superfamily. It presumably plays important roles in controlling the intracellular level of ADP-ribose (ADPR) to prevent non-enzymatic ADP-ribosylation by hydrolyzing ADPR to AMP and ribose 5'-phosphate. We report here the crystal structures of hNUDT5 in apo form, in complex with ADPR, and in complex with AMP with bound Mg2+. hNUDT5 forms a homodimer with substantial domain swapping and assumes a structure more similar to Escherichia coli ADPRase ORF209 than human ADPRase NUDT9. The adenine moiety of the substrates is specifically recognized by the enzyme via hydrogen-bonding interactions between N1 and N6 of the base and Glu47 of one subunit, and between N7 of the base and Arg51 of the other subunit, providing the molecular basis for the high selectivity of hNUDT5 for ADP-sugars over other sugar nucleotides. Structural comparisons with E. coli ADPRase ORF209 and ADPXase ORF186 indicate that the existence of an aromatic residue on loop L8 in ORF186 seems to be positively correlated with its enzymatic activity on APnA, whereas hNUDT5 and ORF209 contain no such residue and thus have low or no activities on APnA.     

Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Adhesive complexes composed of microneme proteins (MICs) are secreted onto the parasite surface from intracellular stores and fulfil crucial roles in host-cell recognition, attachment and penetration. Here, we report the high-resolution solution structure of a complex between two crucial MICs, TgMIC6 and TgMIC1. Furthermore, we identify two analogous interaction sites within separate epidermal growth factor-like (EGF) domains of TgMIC6-EGF2 and EGF3-and confirm that both interactions are functional for the recognition of host cell receptor in the parasite, using immunofluorescence and invasion assays. The nature of this new mode of recognition of the EGF domain and its abundance in apicomplexan surface proteins suggest a more generalized means of constructing functional assemblies by using EGF domains with highly specific receptor-binding properties.     

Structural insights into the regulation of PDK1 by phosphoinositides and inositol phosphates

3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates many kinases belonging to the AGC subfamily. PDK1 possesses a C-terminal pleckstrin homology (PH) domain that interacts with PtdIns(3,4,5)P3/PtdIns(3,4)P2 and with lower affinity to PtdIns(4,5)P2. We describe the crystal structure of the PDK1 PH domain, in the absence and presence of PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4. The structures reveal a 'budded' PH domain fold, possessing an N-terminal extension forming an integral part of the overall fold, and display an unusually spacious ligand-binding site. Mutagenesis and lipid-binding studies were used to define the contribution of residues involved in phosphoinositide binding. Using a novel quantitative binding assay, we found that Ins(1,3,4,5,6)P5 and InsP6, which are present at micromolar levels in the cytosol, interact with full-length PDK1 with nanomolar affinities. Utilising the isolated PDK1 PH domain, which has reduced affinity for Ins(1,3,4,5,6)P5/InsP6, we perform localisation studies that suggest that these inositol phosphates serve to anchor a portion of cellular PDK1 in the cytosol, where it could activate its substrates such as p70 S6-kinase and p90 ribosomal S6 kinase that do not interact with phosphoinositides.     

Analysis of a new crystal form of procarboxypeptidase B: Further insights into the catalytic mechanism

A new triclinic crystal structure form of porcine pancreatic procarboxypeptidase B (PCPB) was obtained at higher resolution than the previously known tetragonal crystal structure. This new crystal polymorph has allowed for a corrected, accurate assignment of residues along the polypeptide chain based on the currently available gene sequence information and crystallographic data. The present structure shows unbound PCPB in a distinct molecular packing as compared to the previous benzamidine complexed form. Its catalytically important Tyr248 residue is oriented and hydrogen‐bonded to solvent water molecules, and locates the furthest away from the catalytic zinc ion as compared to previous structures. A relatively long stretch of residues flanking Tyr248 and guarding the access to the catalytic zinc ion was found to be sequentially unique to the M14 family of peptidases. Predictions from a normal mode analysis indicated that this stretch of residues belongs to a rigid subdomain in the protein structure. The specific presence of a tyrosyl residue at the most exposed position in this region would allow for a delicate balance between extreme hydrophobicity and hydrophilicity, and affect substrate binding and the kinetic efficiency of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 178–185, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The crystal structures of phosphopantetheine adenylyltransferase with bound substrates reveal the enzyme's catalytic mechanism.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme in the coenzyme A pathway that catalyzes the reversible transfer of an adenylyl group from ATP to 4'-phosphopantetheine (Ppant) in the presence of magnesium. To investigate the reaction mechanism, the high-resolution crystal structures of the Escherichia coli PPAT have been determined in the presence of either ATP or Ppant. Structural details of the catalytic center revealed specific roles for individual amino acid residues involved in substrate binding and catalysis. The side-chain of His18 stabilizes the expected pentacovalent intermediate, whereas the side-chains of Thr10 and Lys42 orient the nucleophile for an in-line displacement mechanism. The binding site for the manganese ion that interacts with the phosphate groups of the nucleotide has also been identified. Within the PPAT hexamer, one trimer is in its substrate-free state, whereas the other is in a substrate-bound state.