Kap Lim

Structure of the YibK methyltransferase from Haemophilus influenzae (HI0766): A cofactor bound at a site formed by a knot

The crystal structures of YibK from Haemophilus influenzae (HI0766) have been determined with and without bound cofactor product S‐adenosylhomocysteine (AdoHcy) at 1.7 and 2.0 Å resolution, respectively. The molecule adopts an α/β fold, with a topology that differs from that of the classical methyltransferases. Most notably, HI0766 contains a striking knot that forms the binding crevice for the cofactor. The knot formation is correlated with an alternative arrangement of the secondary structure units compared with the classical methyltransferases. Two loop regions undergo conformational changes upon AdoHcy binding. In contrast to the extended conformation of the cofactor seen in the classical methyltransferase structures, AdoHcy binds to HI0766 in a bent conformation. HI0766 and its close sequence relatives are all shorter versions of the more remotely related rRNA/tRNA methyltransferases of the spoU sequence family. We propose that the spoU sequence family contains the same core domain for cofactor binding as HI0766 but has an additional domain for substrate binding. The substrate‐binding domain is absent in HI0766 sequence family and may be provided by another Haemophilus influenzae partner protein, which is yet to be identified. Proteins 2003;51:56–67.

Structure of phosphorylated enzyme I, the phosphoenolpyruvate:sugar phosphotransferase system sugar translocation signal protein.

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP):sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the Mg2+-dependent phosphorylation of enzyme I (EI) by PEP. Crystals of Escherichia coli EI were obtained by mixing the protein with Mg2+ and PEP, followed by oxalate, an EI inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by Mg2+ and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189∼P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an α-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an EI fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189∼P and His-15 are appropriately stationed for an in-line phosphotransfer reaction.

From structure to function: YrbI from Haemophilus influenzae (HI1679) is a phosphatase

The crystal structure of the YrbI protein from Haemophilus influenzae (HI1679) was determined at a 1.67‐Å resolution. The function of the protein had not been assigned previously, and it is annotated as hypothetical in sequence databases. The protein exhibits the α/β‐hydrolase fold (also termed the Rossmann fold) and resembles most closely the fold of the L‐2‐haloacid dehalogenase (HAD) superfamily. Following this observation, a detailed sequence analysis revealed remote homology to two members of the HAD superfamily, the P‐domain of Ca2+ ATPase and phosphoserine phosphatase. The 19‐kDa chains of HI1679 form a tetramer both in solution and in the crystalline form. The four monomers are arranged in a ring such that four β‐hairpin loops, each inserted after the first β‐strand of the core α/β‐fold, form an eight‐stranded barrel at the center of the assembly. Four active sites are located at the subunit interfaces. Each active site is occupied by a cobalt ion, a metal used for crystallization. The cobalt is octahedrally coordinated to two aspartate side‐chains, a backbone oxygen, and three solvent molecules, indicating that the physiological metal may be magnesium. HI1679 hydrolyzes a number of phosphates, including 6‐phosphogluconate and phosphotyrosine, suggesting that it functions as a phosphatase in vivo. The physiological substrate is yet to be identified; however the location of the gene on the yrb operon suggests involvement in sugar metabolism. Proteins 2002;46:393–404.

Crystal structure of Yeco from Haemophilus influenzae (HI0319) reveals a methyltransferase fold and a bound S‐adenosylhomocysteine

The crystal structure of YecO from Haemophilus influenzae (HI0319), a protein annotated in the sequence databases as hypothetical, and that has not been assigned a function, has been determined at 2.2‐Å resolution. The structure reveals a fold typical of S‐adenosyl‐L‐methionine‐dependent (AdoMet) methyltransferase enzymes. Moreover, a processed cofactor, S‐adenosyl‐L‐homocysteine (AdoHcy), is bound to the enzyme, further confirming the biochemical function of HI0319 and its sequence family members. An active site arginine, shielded from bulk solvent, interacts with an anion, possibly a chloride ion, which in turn interacts with the sulfur atom of AdoHcy. The AdoHcy and nearby protein residues delineate a small solvent‐excluded substrate binding cavity of 162 Å3 in volume. The environment surrounding the cavity indicates that the substrate molecule contains a hydrophobic moiety and an anionic group. Many of the residues that define the cavity are invariant in the HI0319 sequence family but are not conserved in other methyltransferases. Therefore, the substrate specificity of YecO enzymes is unique and differs from the substrate specificity of all other methyltransferases sequenced to date. Examination of the Enzyme Commission list of methyltransferases prompted a manual inspection of 10 possible substrates using computer graphics and suggested that the ortho‐substituted benzoic acids fit best in the active site. Proteins 2001;45:397–407.

Crystal structure of YbaB from Haemophilus influenzae (HI0442), a protein of unknown function coexpressed with the recombinational DNA repair protein RecR

Kap Lim, Aleksandra Tempczyk, James F. Parsons, Nicklas Bonander, John Toedt, Zvi Kelman, Andrew Howard, Edward Eisenstein, and Osnat Herzberg* Center for Advanced Research In Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland National Institute of Standards and Technology, Gaithersburg, Maryland Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois Biological, Chemical, and Physical Science Department, Illinois Institute of Technology, Chicago, Illinois Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland

Structure of 2C-methyl-D-erythrol-2,4-cyclodiphosphate synthase from Haemophilus influenzae: Activation by conformational transition

Christopher Lehmann, Kap Lim, John Toedt, Wojciech Krajewski, Andrew Howard, Edward Eisenstein, and Osnat Herzberg* Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland The National Institute of Standards and Technology, Gaithersburg, Maryland Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois Illinois Institute of Technology, Chicago, Illinois

Crystal structure of the YgfY from Escherichia coli, a protein that may be involved in transcriptional regulation

Introduction. The ygfY gene from Escherichia coli encodes an 88 amino acid protein of unknown function, and is a member of a large sequence family present in both, prokaryotes and eukaryotes. A Psi-Blast search of the nonredundant and environmental nonredundant databases revealed 249 family members. 1 Of these sequences, 152 are from environmental samples, mostly from a Sargasso Sea sample filtered to include only microbial cells. 2 The first cycles of Psi-Blast iteration yielded 78 homologous bacterial proteins, including 42 from the Sargasso Sea. Plant and fungal proteins emerged in the second cycle, and insect and mammalian sequences emerged in the third cycle. While the bacterial proteins have approximately the same size as YgfY (except for incomplete sequences from environmental samples), the eukaryotic proteins are at least double in size. None of the sequence homologues has known biochemical function. The cellular role is also unknown, except for a remote relationship (E-score 10 4 ) to a protein from Saccharomyces cerevisiae, YOL071W/EM15 (GI:6324501), which is required for sporulation and for transcriptional induction of the early meiotic-specific transcription factor IME1. 3 This 162amino-acid residue yeast protein exhibits 25% identity to YgfY over a stretch of 60 amino acids. The crystal structure of YgfY was determined at 1.2 A ˚ resolution as a part of our structural genomics project (http://s2f.umbi.umd.edu), revealing a five-helix fold similar to that of the homologous protein NMA1147 from Neisseria meningitidis (30% identity) determined recently by NMR methods. 4 Yet, there are inconsistencies in the details of these two structures that reflect the different levels of accuracy of the two methods of structure determination. We propose that the functional region is different from the one proposed based on the NMR structure, and highlight a particularly important structural difference associated with the proposed activity center. Materials and Methods. YgfY from E. coli was amplified using PfuTurbo DNA polymerase (Stratagene), genomic DNA, and 5- and 3-end primers. In addition to the native gene sequence, a sequence consistent with a thrombin cleavage site was introduced, and a NdeI restriction site was designed to convert the 6xHis-tagged construct into a construct coding for native protein. The PCR product was introduced into the pET100/D-TOPO expression vector by TOPO directional cloning procedure (Invitrogen). For wildtype protein production, the E. coli strain BL21 Star (DE3) was transformed with the recombinant plasmid. An expression screen showed that the His-tagged protein was soluble, whereas the native protein was insoluble. Wild-type protein was produced by growing the cells at 37°C in Super Broth medium supplemented by ampicillin (100 g/mL). Once the cell culture reached A600 0.6, expression was induced by the addition of 1 mM IPTG, and afte r3ht he cells were harvested by centrifugation. To prepare selenomethionine (SeMet) containing protein, the E. coli B834 (DE3) strain was transformed with the recombinant vector. The cells were grown at 30°C in minimal medium supplemented with ampicillin (50 g/mL), SeMet, and 19 amino acids other than methionine. When the cell culture reached A600 0.5, 1 mM IPTG was added, and after 3 h the cells were harvested. Cells were suspended in 20 mM Tris HCl (pH 8.0), 0.5 M NaCl, and 5 mM imidazole, and lysed by passage through a French press. The soluble fraction was loaded on Ni-NTA metal affinity column (Qiagen). Protein was eluted with 20 mM Tris HCl (pH 8.0), 0.5 M NaCl, and 250 mM imidazole. To remove the N-terminal sequence containing the 6xHis tag, human thrombin (Haemtech) was added at 1:2000 molar ratio and incubated overnight at 4°C in Tris HCl (pH 8.0), and 500 mM NaCl. Thrombin was removed by passing the protein mixture through benzamidine column (Amersham Biosciences). The cleaved and uncleaved protein and the N-terminal peptide were separated on a second Ni-NTA column. The protein was dialyzed against a buffer of 50 mM NaCl and 20 mM Tris HCl (pH 7.5), and further purified with a size-exclusion chromatography

Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member.

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg(2+)-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, alpha and beta, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GKbeta encodes a peptide that is different in sequence and is 16 amino acids longer than that encoded by the N-terminal exon of GKalpha. The crystal structures of recombinant GKalphabeta and GKbetabeta from Namalycastis sp. were determined at 2.6 and 2.4 A resolution, respectively. In addition, the structure of the GKbetabeta was determined at 2.3 A resolution in complex with a transition state analogue, Mg(2+)-ADP-NO(3)(-)-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GKalphabeta are structurally defined, and the longer N-terminus of the beta subunit is anchored at the dimer interface. In GKbetabeta the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GKalphabeta amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely alpha or solely beta chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Background: Carbamate kinase is an essential Giardia lamblia enzyme, and the anti-alcoholism drug disulfiram kills the trophozoites and inhibits the enzyme. Results: Disulfiram acts by modifying Cys-242 adjacent to the active site and cures giardiasis in mice. Conclusion: G. lamblia CK is a good drug target and disulfiram may be repurposed as antigiardiasis drug. Significance: We need new antigiardiasis drugs because current treatments fail frequently. Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC50 values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.

The HI0073/HI0074 protein pair from Haemophilus influenzae is a member of a new nucleotidyltransferase family: Structure, sequence analyses, and solution studies

The crystal structure of HI0074 from Haemophilus influenzae, a protein of unknown function, has been determined at a resolution of 2.4 Å. The molecules form an up–down, four‐helix bundle, and associate into homodimers. The fold is most closely related to the substrate‐binding domain of KNTase, yet the amino acid sequences of the two proteins exhibit no significant homology. Sequence analyses of completely and incompletely sequenced genomes reveal that the two adjacent genes, HI0074 and HI0073, and their close relatives comprise a new family of nucleotidyltransferases, with 15 members at the time of writing. The analyses also indicate that this is one of eight families of a large nucleotidyltransferase superfamily, whose members were identified based on the proximity of the nucleotide‐ and substrate‐binding domains on the respective genomes. Both HI0073 and HI0074 were annotated “hypothetical” in the original genome sequencing publication. HI0073 was cloned, expressed, and purified, and was shown to form a complex with HI0074 by polyacrylamide gel electrophoresis under nondenaturing conditions, analytic size exclusion chromatography, and dynamic light scattering. Double‐ and single‐stranded DNA binding assays showed no evidence of DNA binding to HI0074 or to HI0073/HI0074 complex despite the suggestive shape of the putative binding cleft formed by the HI0074 dimer. Proteins 2003;50:249–260.