The Allosteric Regulation of Β-Ureidopropionase Depends on Fine-Tuned Stability of Active-Site Loops and Subunit Interfaces.

The activity of ß-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human ß-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human ß-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.

The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

CCP4 Cloud for structure determination and project management in macromolecular crystallography.

Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability. There is also an issue of long-term data maintenance and availability of structure-solution projects as the links between experimental observations and the final results deposited in the PDB. In this article, CCP4 Cloud, a new front-end of the CCP4 software suite, is presented which mitigates these effects by providing an online, cloud-based environment for crystallographic computation. CCP4 Cloud was developed for the efficient delivery of computing power, database services and seamless integration with web resources. It provides a rich graphical user interface that allows project sharing and long-term storage for structure-solution projects, and can be linked to data-producing facilities. The system is distributed with the CCP4 software suite version 7.1 and higher, and an online publicly available instance of CCP4 Cloud is provided by CCP4.

Missense variants in DPYSL5 cause a neurodevelopmental disorder with corpus callosum agenesis and cerebellar abnormalities.

The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and ßIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and ßIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.

Current developments in Coot for macromolecular model building of Electron Cryo-microscopy and Crystallographic Data.

Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo-EM model building and refinement, as cryo-EM structures with resolution ranging 2.5-4 A are now routinely available. Model building into these maps can be time-consuming and requires experience in both biochemistry and building into low-resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman-McClure restraints, full-chain refinement, and Fourier-model based residue-type-specific Ramachandran restraints. Here, we present the current state-of-the-art in Coot usage.

Crystal structure and pH-dependent allosteric regulation of human ß-ureidopropionase, an enzyme involved in anticancer drug metabolism.

ß-Ureidopropionase (ßUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like ßUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N-carbamoyl-ß-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human ßUP is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as a homodimer at pH 9, the enzyme increasingly associates to form octamers and larger oligomers with decreasing pH. Only at physiological pH is the enzyme responsive to effector binding, with N-carbamoyl-ß-alanine causing association to more active higher molecular mass species, and ß-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests that protonation state changes play a crucial role in the allosteric regulation mechanism. Disruption of dimer-dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Šresolution revealed high structural conservation between human and fruit fly ßUP, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer-dimer interface, effectively positioning the catalytically important Glu207 in the active site.

Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone.

Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine- N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine- N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.

Ab initio solution of macromolecular crystal structures without direct methods.

The majority of macromolecular crystal structures are determined using the method of molecular replacement, in which known related structures are rotated and translated to provide an initial atomic model for the new structure. A theoretical understanding of the signal-to-noise ratio in likelihood-based molecular replacement searches has been developed to account for the influence of model quality and completeness, as well as the resolution of the diffraction data. Here we show that, contrary to current belief, molecular replacement need not be restricted to the use of models comprising a substantial fraction of the unknown structure. Instead, likelihood-based methods allow a continuum of applications depending predictably on the quality of the model and the resolution of the data. Unexpectedly, our understanding of the signal-to-noise ratio in molecular replacement leads to the finding that, with data to sufficiently high resolution, fragments as small as single atoms of elements usually found in proteins can yield ab initio solutions of macromolecular structures, including some that elude traditional direct methods.

Crystal structures of the kinase domain of the sulfate-activating complex in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis the sulfate activating complex provides a key branching point in sulfate assimilation. The complex consists of two polypeptide chains, CysD and CysN. CysD is an ATP sulfurylase that, with the energy provided by the GTPase activity of CysN, forms adenosine-5'-phosphosulfate (APS) which can then enter the reductive branch of sulfate assimilation leading to the biosynthesis of cysteine. The CysN polypeptide chain also contains an APS kinase domain (CysC) that phosphorylates APS leading to 3'-phosphoadenosine-5'-phosphosulfate, the sulfate donor in the synthesis of sulfolipids. We have determined the crystal structures of CysC from M. tuberculosis as a binary complex with ADP, and as ternary complexes with ADP and APS and the ATP mimic AMP-PNP and APS, respectively, to resolutions of 1.5 Å, 2.1 Å and 1.7 Å, respectively. CysC shows the typical APS kinase fold, and the structures provide comprehensive views of the catalytic machinery, conserved in this enzyme family. Comparison to the structure of the human homolog show highly conserved APS and ATP binding sites, questioning the feasibility of the design of specific inhibitors of mycobacterial CysC. Residue Cys556 is part of the flexible lid region that closes off the active site upon substrate binding. Mutational analysis revealed this residue as one of the determinants controlling lid closure and hence binding of the nucleotide substrate.

Crystal structure of human CRMP-4: correction of intensities for lattice-translocation disorder.

Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins that are mainly involved in neuronal cell development. In humans, the CRMP family comprises five members. Here, crystal structures of human CRMP-4 in a truncated and a full-length version are presented. The latter was determined from two types of crystals, which were either twinned or partially disordered. The crystal disorder was coupled with translational NCS in ordered domains and manifested itself with a rather sophisticated modulation of intensities. The data were demodulated using either the two-lattice treatment of lattice-translocation effects or a novel method in which demodulation was achieved by independent scaling of several groups of intensities. This iterative protocol does not rely on any particular parameterization of the modulation coefficients, but uses the current refined structure as a reference. The best results in terms of R factors and map correlation coefficients were obtained using this new method. The determined structures of CRMP-4 are similar to those of other CRMPs. Structural comparison allowed the confirmation of known residues, as well as the identification of new residues, that are important for the homo- and hetero-oligomerization of these proteins, which are critical to nerve-cell development. The structures provide further insight into the effects of medically relevant mutations of the DPYSL-3 gene encoding CRMP-4 and the putative enzymatic activities of CRMPs.

Substrate Channel Flexibility in Pseudomonas aeruginosa MurB Accommodates Two Distinct Substrates.

Biosynthesis of UDP-N-acetylmuramic acid in bacteria is a committed step towards peptidoglycan production. In an NADPH- and FAD-dependent reaction, the UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) reduces UDP-N-acetylglucosamine-enolpyruvate to UDP-N-acetylmuramic acid. We determined the three-dimensional structures of the ternary complex of Pseudomonas aeruginosa MurB with FAD and NADP(+) in two crystal forms to resolutions of 2.2 and 2.1 Å, respectively, to investigate the structural basis of the first half-reaction, hydride transfer from NADPH to FAD. The nicotinamide ring of NADP(+) stacks against the si face of the isoalloxazine ring of FAD, suggesting an unusual mode of hydride transfer to flavin. Comparison with the structure of the Escherichia coli MurB complex with UDP-N-acetylglucosamine-enolpyruvate shows that both substrates share the binding site located between two lobes of the substrate-binding domain III, consistent with a ping pong mechanism with sequential substrate binding. The nicotinamide and the enolpyruvyl moieties are strikingly well-aligned upon superimposition, both positioned for hydride transfer to and from FAD. However, flexibility of the substrate channel allows the non-reactive parts of the two substrates to bind in different conformations. A potassium ion in the active site may assist in substrate orientation and binding. These structural models should help in structure-aided drug design against MurB, which is essential for cell wall biogenesis and hence bacterial survival.

Insights into the oligomerization of CRMPs: crystal structure of human collapsin response mediator protein 5.

Collapsin response mediator protein-5 (CRMP-5) is the latest identified member of the CRMP cytosolic phosphoprotein family, which is crucial for neuronal development and repair. CRMPs exist as homo- and/or hetero-tetramers in vivo and participate in signaling transduction, cytoskeleton rearrangements, and endocytosis. CRMP-5 antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners. We determined the crystal structures of a full length and a truncated version of human CRMP-5, both of which form a homo-tetramer similar to those observed in CRMP-1 and CRMP-2. However, solution studies indicate that CRMP-5 and CRMP-1 form weaker homo-tetramers compared with CRMP-2, and that divalent cations, Ca(2+) and Mg(2+), destabilize oligomers of CRMP-5 and CRMP-1, but promote CRMP-2 oligomerization. On the basis of comparative analysis of the CRMP-5 crystal structure, we identified residues that are crucial for determining the preference for hetero-oligomer or homo-oligomer formation. We also show that in spite of being the CRMP family member most closely related to dihydropyrimidinase, CRMP-5 does not have any detectable amidohydrolase activity. The presented findings provide new detailed insights into the structure, oligomerization, and regulation of CRMPs.

Novel INF2 mutation p. L77P in a family with glomerulopathy and Charcot-Marie-Tooth neuropathy.

BACKGROUND: Mutations in inverted formin, FH2, and WH2 domain containing (INF2) are common causes of dominant focal segmental glomerulosclerosis. INF2 encodes a member of the diaphanous-related formin family, which regulates actin and microtubule cytoskeletons. Charcot-Marie-Tooth neuropathy (CMT) is a group of inherited disorders affecting peripheral neurons. Many reports have shown that glomerulopathy can associate with CMT. However, it has been unclear whether these two processes in the same individual represent one disorder or if they are two separate diseases. CASE DIAGNOSIS/TREATMENT: Recently, INF2 mutations were identified in 12 of 16 patients with CMT-associated glomerulopathy, suggesting that these mutations are a common cause of the dual phenotype. In this study, we report two cases of CMT-associated glomerulopathy that both showed INF2 mutations. A novel INF2 mutation, p. L77P, was identified in a family in which the dual phenotype was inherited in a dominant fashion. The pathogenic effect of p. L77P was proposed using a structural homology model. In addition, we identified a patient with a sporadic CMT-associated glomerulopathy carrying a known INF2 mutation: p. L128P. CONCLUSIONS: Our study confirms the link between INF2 mutations and CMT-associated glomerulopathy and widens the spectrum of pathogenic mutations.

ß-ureidopropionase deficiency: phenotype, genotype and protein structural consequences in 16 patients.

ß-ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO(2). To date, only five genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified ß-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding ß-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant ß-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ß-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated.

Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination.

In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.

A mixture of fortunes: the curious determination of the structure of Escherichia coli BL21 Gab protein.

In protein crystallography, monodisperse protein samples of high purity are usually required in order to obtain diffraction-quality crystals. Here, crystals were reproducibly grown from a protein sample before its homogeneity had been determined. The sample was obtained after the first attempt to purify a recombinant target protein from an Escherichia coli cell lysate. Subsequent analysis revealed that it was a mixture of about 50 different proteins with no predominant species. Diffraction data were collected to 2.1 A and the space group was identified as I422. A molecular-replacement search with models of the expected target did not give a solution, which suggested that a contaminating E. coli protein had been crystallized. A PDB search revealed 256 structures determined in space group I422, of which 14 are E. coli proteins and two have unit-cell parameters similar to those observed. Molecular replacement with these structures showed a clear solution for one of them, the Gab protein. The structure is presented and compared with the deposited structure, from which it shows small but significant differences. The refined model contains bicine and sulfate as bound ligands, which provide insights into possible substrate-binding sites.

The crystal structure of beta-alanine synthase from Drosophila melanogaster reveals a homooctameric helical turn-like assembly.

Beta-alanine synthase (betaAS) is the third enzyme in the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of the nucleotide bases uracil and thymine in higher organisms. It catalyzes the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate to the corresponding beta-amino acids. betaASs are grouped into two phylogenetically unrelated subfamilies, a general eukaryote one and a fungal one. To reveal the molecular architecture and understand the catalytic mechanism of the general eukaryote betaAS subfamily, we determined the crystal structure of Drosophila melanogaster betaAS to 2.8 A resolution. It shows a homooctameric assembly of the enzyme in the shape of a left-handed helical turn, in which tightly packed dimeric units are related by 2-fold symmetry. Such an assembly would allow formation of higher oligomers by attachment of additional dimers on both ends. The subunit has a nitrilase-like fold and consists of a central beta-sandwich with a layer of alpha-helices packed against both sides. However, the core fold of the nitrilase superfamily enzymes is extended in D. melanogaster betaAS by addition of several secondary structure elements at the N-terminus. The active site can be accessed from the solvent by a narrow channel and contains the triad of catalytic residues (Cys, Glu, and Lys) conserved in nitrilase-like enzymes.

Clinical, biochemical and genetic findings in two siblings with a dihydropyrimidinase deficiency.

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and it catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine to N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid, respectively. To date, only nine individuals have been reported suffering from a complete DHP deficiency. We report two siblings presenting with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. One of the siblings had a severe delay in speech development and white matter abnormalities, whereas the other one was free of symptoms. Analysis of the DHP gene (DPYS) showed that both patients were compound heterozygous for the missense mutation 1078T>C (W360R) in exon 6 and a novel missense mutation 1235G>T (R412M) in exon 7. Heterologous expression of the mutant enzymes in Escherichia coli showed that both missense mutations resulted in a mutant DHP enzyme without residual activity. Analysis of the crystal structure of eukaryotic DHP from the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum suggests that the W360R and R412M mutations lead to structural instability of the enzyme which could potentially impair the assembly of the tetramer.

Three-dimensional structure of iminodisuccinate epimerase defines the fold of the MmgE/PrpD protein family.

Iminodisuccinate (IDS) epimerase catalyzes the epimerisation of R,R-, S,S- and R,S- iminodisuccinate, one step in the biodegradation of the chelating agent iminodisuccinate by Agrobacterium tumefaciens BY6. The enzyme is a member of the MmgE/PrpD protein family, a diverse and little characterized class of proteins of prokaryotic and eukaryotic origin. IDS epimerase does not show significant overall amino acid sequence similarity to any other protein of known three-dimensional structure. The crystal structure of this novel epimerase has been determined by multi-wavelength diffraction to 1.5 A resolution using selenomethionine-substituted enzyme. In the crystal, the enzyme forms a homo-dimer, and the subunit consists of two domains. The larger domain, not consecutive in sequence and comprising residues Met1-Lys266 and Leu400-Pro446, forms a novel all alpha-helical fold with a central six-helical bundle. The second, smaller domain folds into an alpha+beta domain, related in topology to chorismate mutase by a circular permutation. IDS epimerase is thus not related in three-dimensional structure to other known epimerases. The fold of the IDS epimerase is representative for the whole MmgE/PrpD family. The putative active site is located at the interface between the two domains of the subunit, and is characterized by a positively charged surface, consistent with the binding of a highly negatively charged substrate such as iminodisuccinate. Docking experiments suggest a two-base mechanism for the epimerisation reaction.

Crystal structures of SnoaL2 and AclR: two putative hydroxylases in the biosynthesis of aromatic polyketide antibiotics.

SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.