J.R.C. Muniz

Structures of the Human GTPase MMAA and Vitamin B12-dependent Methylmalonyl-CoA Mutase and Insight into Their Complex Formation

Vitamin B12 (cobalamin, Cbl) is essential to the function of two human enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MUT). The conversion of dietary Cbl to its cofactor forms, methyl-Cbl (MeCbl) for MS and adenosyl-Cbl (AdoCbl) for MUT, located in the cytosol and mitochondria, respectively, requires a complex pathway of intracellular processing and trafficking. One of the processing proteins, MMAA (methylmalonic aciduria type A), is implicated in the mitochondrial assembly of AdoCbl into MUT and is defective in children from the cblA complementation group of cobalamin disorders. To characterize the functional interplay between MMAA and MUT, we have crystallized human MMAA in the GDP-bound form and human MUT in the apo, holo, and substrate-bound ternary forms. Structures of both proteins reveal highly conserved domain architecture and catalytic machinery for ligand binding, yet they show substantially different dimeric assembly and interaction, compared with their bacterial counterparts. We show that MMAA exhibits GTPase activity that is modulated by MUT and that the two proteins interact in vitro and in vivo. Formation of a stable MMAA-MUT complex is nucleotide-selective for MMAA (GMPPNP over GDP) and apoenzyme-dependent for MUT. The physiological importance of this interaction is highlighted by a recently identified homoallelic patient mutation of MMAA, G188R, which, we show, retains basal GTPase activity but has abrogated interaction. Together, our data point to a gatekeeping role for MMAA by favoring complex formation with MUT apoenzyme for AdoCbl assembly and releasing the AdoCbl-loaded holoenzyme from the complex, in a GTP-dependent manner.

Targeting Low-Druggability Bromodomains: Fragment Based Screening and Inhibitor Design against the BAZ2B Bromodomain

Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable.

Ribosomal Oxygenases are Structurally Conserved from Prokaryotes to Humans.

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

Molecular mechanism of peroxisome proliferator-activated receptor α activation by WY14643 : a new mode of ligand recognition and receptor stabilization

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys189 is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from PII SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu276 of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other PIII SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other PIII members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis.

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site‐directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF‐5A protein‐folding defects. Our data provided evidence that the C‐terminal α‐helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N‐terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature‐sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature‐sensitive strains produced stable mutant eIF5A proteins – eIF5AK56A and eIF5AQ22H,L93F– and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Joint X‐ray crystallographic and molecular dynamics study of cellobiohydrolase I from Trichoderma harzianum: deciphering the structural features of cellobiohydrolase catalytic activity

Aiming to contribute toward the characterization of new, biotechnologically relevant cellulolytic enzymes, we report here the first crystal structure of the catalytic core domain of Cel7A (cellobiohydrolase I) from the filamentous fungus Trichoderma harzianum IOC 3844. Our structural studies and molecular dynamics simulations show that the flexibility of Tyr260, in comparison with Tyr247 from the homologous Trichoderma reesei Cel7A, is enhanced as a result of the short side‐chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. T. harzianum cellobiohydrolase I also has a shortened loop at the entrance of the cellulose‐binding tunnel, which has been described to interact with the substrate in T. reesei Cel7A. These structural features might explain why T. harzianum Cel7A displays higher kcat and Km values, and lower product inhibition on both glucoside and lactoside substrates, compared with T. reesei Cel7A.

Crystal structure of human carbonic anhydrase-related protein VIII reveals the basis for catalytic silencing.

Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton.1 They are ubiquitous in prokaryotes and eukaryotes, and are encoded by four evolutionarily-unrelated gene classes (a, b, g, d). The human CA family belongs to the a class and contains 12 catalytic isozymes with different tissue distribution, subcellular localization and kinetic properties. These include the cytosolic (CA I, CA II, CA III, CA VII, CA XIII), membrane-bound (CA IV, CA IX, CA XII, CA XIV), mitochondrial (CA VA, CA VB) and secreted (CA VI) isoforms. They share an absolute requirement for a catalytic Zn ion in the active site, coordinated by an hydroxide ion and three invariant histidine residues H94CAII, H96CAII and H119CAII (human CA II numbering) that are in turn hydrogen bonded to conserved partners Q92CAII, N244CAII and E117CAII, respectively. CAs participate in physiological processes such as respiration, metabolite biosynthesis and pH regulation, and are interesting pharmaceutical targets.2 Sulphonamide-based CA inhibitors are established diuretics and anti-glaucoma drugs, and may have further therapeutic potentials in anti-obesity and anti-cancer treatment.3 The human CA family includes a subclass of three noncatalytic isoforms (CA VIII, CA X, CA XI), also known as CA-related proteins (CA-RPs), based on sequence homology with the catalytic isozymes. CA-RPs lack one or more of the essential Zn-coordinating histidines and are devoid of CO2 hydration activity.4 To date, the biological functions of CA-RPs remain undefined. The first identified CA-RP, CA VIII, replaces the Zn-coordinating H94CAII and its hydrogen-bonding partner Q92CAII with R116 and E114, respectively (human CA VIII numbering).5 CA VIII is highly expressed in the cerebellum,6 and a mouse gene deletion causes a motor coordination defect.7 Relevant to this, CA VIII has been identified as a binding partner for the inositol 1,4,5 triphosphate (IP3) receptor type 1 which is abundant in the cerebellum.8 To provide insights into the biological properties of CA-RPs, we have determined the 1.6 A crystal structure of human CA VIII (hCA VIII). This work represents the first structural characterization of a CA-RP and offers a structural basis for its catalytic silencing effect.

Insight into S-adenosylmethionine biosynthesis from the crystal structures of the human methionine adenosyltransferase catalytic and regulatory subunits

MAT (methionine adenosyltransferase) utilizes L-methionine and ATP to form SAM (S-adenosylmethionine), the principal methyl donor in biological methylation. Mammals encode a liver-specific isoenzyme, MAT1A, that is genetically linked with an inborn metabolic disorder of hypermethioninaemia, as well as a ubiquitously expressed isoenzyme, MAT2A, whose enzymatic activity is regulated by an associated subunit MAT2B. To understand the molecular mechanism of MAT functions and interactions, we have crystallized the ligand-bound complexes of human MAT1A, MAT2A and MAT2B. The structures of MAT1A and MAT2A in binary complexes with their product SAM allow for a comparison with the Escherichia coli and rat structures. This facilitates the understanding of the different substrate or product conformations, mediated by the neighbouring gating loop, which can be accommodated by the compact active site during catalysis. The structure of MAT2B reveals an SDR (short-chain dehydrogenase/reductase) core with specificity for the NADP/H cofactor, and harbours the SDR catalytic triad (YxxxKS). Extended from the MAT2B core is a second domain with homology with an SDR sub-family that binds nucleotide-sugar substrates, although the equivalent region in MAT2B presents a more open and extended surface which may endow a different ligand/protein-binding capability. Together, the results of the present study provide a framework to assign structural features to the functional and catalytic properties of the human MAT proteins, and facilitate future studies to probe new catalytic and binding functions.

Molecular Architecture of the Ankyrin SOCS Box Family of Cul5-Dependent E3 Ubiquitin Ligases

Multi-subunit Cullin–RING E3 ligases often use repeat domain proteins as substrate-specific adaptors. Structures of these macromolecular assemblies are determined for the F-box-containing leucine-rich repeat and WD40 repeat families, but not for the suppressor of cytokine signaling (SOCS)-box-containing ankyrin repeat proteins (ASB1–18), which assemble with Elongins B and C and Cul5. We determined the crystal structures of the ternary complex of ASB9–Elongin B/C as well as the interacting N-terminal domain of Cul5 and used structural comparisons to establish a model for the complete Cul5-based E3 ligase. The structures reveal a distinct architecture of the ASB9 complex that positions the ankyrin domain coaxial to the SOCS box–Elongin B/C complex and perpendicular to other repeat protein complexes. This alternative architecture appears favorable to present the ankyrin domain substrate-binding site to the E2-ubiquitin, while also providing spacing suitable for bulky ASB9 substrates, such as the creatine kinases. The presented Cul5 structure also differs from previous models and deviates from other Cullins via a rigid-body rotation between Cullin repeats. This work highlights the adaptability of repeat domain proteins as scaffolds in substrate recognition and lays the foundation for future structure–function studies of this important E3 family.