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Sung-Hou Kim

Publications and source records attributed to Sung-Hou Kim.

At least 19 recordsLinked to original sources
PubMed2006

Effect of osmotic stress and heat shock in recombinant protein overexpression and crystallization.

Overexpressed recombinant proteins in bacteria often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or of chaperones. This can be accomplished by growing the bacterial cells in the presence of high salt, sorbitol, and betaine as well as exposing the cells to a heat shock step. Our results suggest that by growing the cells under varied conditions one may be able to express targets as soluble proteins (from previously insoluble targets) and to improve the chances of their crystallization.

Crystallization↗
PubMed2006

Evolution of protein structural classes and protein sequence families.

In protein structure space, protein structures cluster into four elongated regions when mapped based solely on similarity among the 3D structures. These four regions correspond to the four major classes of present-day proteins defined by the contents of secondary structure types and their topological arrangement. Evolution of and restriction to these four classes suggest that, in most cases, the evolution of genes may have been constrained or selected to those genetic changes that results in structurally stable proteins occupying one of the four "allowed" regions of the protein structure space, "structural selection," an important component of natural selection in gene evolution. Our studies on tracing the "common structural ancestor" for each protein sequence family of known structure suggest that: (i) recently emerged proteins belong mostly to three classes; (ii) the proteins that emerged earlier evolved to gain a new class; and (iii) the proteins that emerged earliest evolved to become the present-day proteins in the four major classes, with the fourth-class proteins becoming the most dominant population. Furthermore, our studies also show that not all present-day proteins evolved from one single set of proteins in the last common ancestral organism, but new common ancestral proteins were "born" at different evolutionary times, not traceable to one or two ancestral proteins: "the multiple birth model" for the evolution of protein sequence families.

Evolution, Molecular↗
PubMed2006

Structural proteomics of minimal organisms: conservation of protein fold usage and evolutionary implications.

BACKGROUND: Determining the complete repertoire of protein structures for all soluble, globular proteins in a single organism has been one of the major goals of several structural genomics projects in recent years. RESULTS: We report that this goal has nearly been reached for several "minimal organisms"--parasites or symbionts with reduced genomes--for which over 95% of the soluble, globular proteins may now be assigned folds, overall 3-D backbone structures. We analyze the structures of these proteins as they relate to cellular functions, and compare conservation of fold usage between functional categories. We also compare patterns in the conservation of folds among minimal organisms and those observed between minimal organisms and other bacteria. CONCLUSION: We find that proteins performing essential cellular functions closely related to transcription and translation exhibit a higher degree of conservation in fold usage than proteins in other functional categories. Folds related to transcription and translation functional categories were also overrepresented in minimal organisms compared to other bacteria.

Bacterial Proteins↗
PubMed2006

A method for evaluating the structural quality of protein models by using higher-order phi-psi pairs scoring.

A method is presented for scoring the model quality of experimental and theoretical protein structures. The structural model to be evaluated is dissected into small fragments via a sliding window, where each fragment is represented by a vector of multiple phi-psi angles. The sliding window ranges in size from a length of 1-10 phi-psi pairs (3-12 residues). In this method, the conformation of each fragment is scored based on the fit of multiple phi-psi angles of the fragment to a database of multiple phi-psi angles from high-resolution x-ray crystal structures. We show that measuring the fit of predicted structural models to the allowed conformational space of longer fragments is a significant discriminator for model quality. Reasonable models have higher-order phi-psi score fit values (m) > -1.00.

Models, Molecular↗
PubMed2006

Crystal structure of the DUF16 domain of MPN010 from Mycoplasma pneumoniae.

We have determined the crystal structure of the DUF16 domain of unknown function encoded by the gene MPN010 of Mycoplasma pneumoniae at 1.8 A resolution. The crystal structure revealed that this domain is composed of two separated homotrimeric coiled-coils. The shorter one consists of 11 highly conserved residues. The sequence comprises noncanonical heptad repeats that induce a right-handed coiled-coil structure. The longer one is composed of approximately nine heptad repeats. In this coiled-coil structure, there are three distinguishable regions that confer unique structural properties compared with other known homotrimeric coiled-coils. The first part, containing one stutter, is an unusual phenylalanine-rich region that is not found in any other coiled-coil structures. The second part is a highly conserved glutamine-rich region, frequently found in other trimeric coiled-coil structures. The last part is composed of prototype heptad repeats. The phylogenetic analysis of the DUF16 family together with a secondary structure prediction shows that the DUF16 family can be classified into five subclasses according to N-terminal sequences. Based on the structural comparison with other coiled-coil structures, a probable molecular function of the DUF16 family is discussed.

Amino Acid Sequence↗
PubMed2006

Target selection and deselection at the Berkeley Structural Genomics Center.

At the Berkeley Structural Genomics Center (BSGC), our goal is to obtain a near-complete structural complement of proteins in the minimal organisms Mycoplasma genitalium and M. pneumoniae, two closely related pathogens. Current targets for structure determination have been selected in six major stages, starting with those predicted to be most tractable to high throughput study and likely to yield new structural information. We report on the process used to select these proteins, as well as our target deselection procedure. Target deselection reduces experimental effort by eliminating targets similar to those recently solved by the structural biology community or other centers. We measure the impact of the 69 structures solved at the BSGC as of July 2004 on structure prediction coverage of the M. pneumoniae and M. genitalium proteomes. The number of Mycoplasma proteins for which the fold could first be reliably assigned based on structures solved at the BSGC (24 M. pneumoniae and 21 M. genitalium) is approximately 25% of the total resulting from work at all structural genomics centers and the worldwide structural biology community (94 M. pneumoniae and 86 M. genitalium) during the same period. As the number of structures contributed by the BSGC during that period is less than 1% of the total worldwide output, the benefits of a focused target selection strategy are apparent. If the structures of all current targets were solved, the percentage of M. pneumoniae proteins for which folds could be reliably assigned would increase from approximately 57% (391 of 687) at present to around 80% (550 of 687), and the percentage of the proteome that could be accurately modeled would increase from around 37% (254 of 687) to about 64% (438 of 687). In M. genitalium, the percentage of the proteome that could be structurally annotated based on structures of our remaining targets would rise from 72% (348 of 486) to around 76% (371 of 486), with the percentage of accurately modeled proteins would rise from 50% (243 of 486) to 58% (283 of 486). Sequences and data on experimental progress on our targets are available in the public databases TargetDB and PEPCdb.

Amino Acids↗
PubMed2006

Crystal structure of ScpB from Chlorobium tepidum, a protein involved in chromosome partitioning.

Structural maintenance of chromosome (SMC) proteins are essential in chromosome condensation and interact with non-SMC proteins in eukaryotes and with segregation and condensation proteins (ScpA and ScpB) in prokaryotes. The highly conserved gene in Chlorobium tepidum gi 21646405 encodes ScpB (ScpB_ChTe). The high resolution crystal structure of ScpB_ChTe shows that the monomeric structure consists of two similarly shaped globular domains composed of three helices sided by beta-strands [a winged helix-turn-helix (HTH)], a motif observed in the C-terminal domain of Scc1, a functionally related eukaryotic ScpA homolog, as well as in many DNA binding proteins.

Amino Acid Sequence↗
PubMed2005

Crystal structures of a phosphotransacetylase from Bacillus subtilis and its complex with acetyl phosphate.

Phosphotransacetylase (Pta) [EC 2.3.1.8] plays a major role in acetate metabolism by catalyzing the reversible transfer of the acetyl group between coenzyme A (CoA) and orthophosphate: CH(3)COSCoA+HPO(4)(2-)<-->CH(3)COOPO(3)(2-) +CoASH. In this study, we report the crystal structures of Pta from Bacillus subtilis at 2.75 A resolution and its complex with acetyl phosphate, one of its substrates, at 2.85 A resolution. In addition, the Pta activity of the enzyme has been assayed. The enzyme folds into an alpha/beta architecture with two domains separated by a prominent cleft, very similar to two other known Pta structures. The enzyme-acetyl phosphate complex structure reveals a few potential substrate binding sites. Two of them are located in the middle of the interdomain cleft: each one is surrounded by a region of strictly and highly conserved residues. High structural similarities are found with 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), and isocitrate and isopropylmalate dehydrogenases, all of which utilize NADP+ as their cofactor, which binds in the interdomain cleft. Their substrate binding sites are close to the acetyl phosphate binding sites of Pta in the cleft as well. These results suggest that the CoA is likely to bind to the interdomain cleft of Pta in a similar way as NADP+ binds to the other three enzymes.

Amino Acid Sequence↗
PubMed2005

Structural and functional characterization of a 5,10-methenyltetrahydrofolate synthetase from Mycoplasma pneumoniae (GI: 13508087).

Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase [MTHFS; also known as 5-formyltetrahydrofolate cycloligase; Enzyme Commission (EC) 6.3.3.2] belongs to a large cycloligase protein family with 97 sequence homologues from bacteria to human. To help define the molecular (biochemical and biophysical) function of the M. pneumoniae MTHFS, we have previously determined its crystal structure at 2.2 A resolution (Chen et al., Proteins 2004;56:839-843). In this current study, activity assays confirmed the functionality of the recombinant protein, with K(m) = 165 microM for 5-formyltetrahydrofolate (5-FTHF) and K(m) = 166 microM for MgATP. The methenyltetrahydrofolate activity of M. pneumoniae MTHFS has a requirement for divalent metal ions with Mg2+ being most effective, and an absolute requirement for nucleoside 5'-triphosphates with adenosine triphosphate (ATP) being most effective. Crystallization in the presence of substrates (MgATP, with or without 5-FTHF) produced the complex structures of the protein with adenosine diphosphate (ADP) and phosphate at 2.2 A resolution; with ADP, phosphate, and 5-FTHF at 2.5 A resolution. These structures directly demonstrated that the role of Mg2+ in the reaction is to form the ATP--Mg2+-enzyme complex.

Adenosine Diphosphate↗
PubMed2005

Crystal structure of TM1457 from Thermotoga maritima.

The crystal structure of a hypothetical protein, TM1457, from Thermotoga maritima has been determined at 2.0A resolution. TM1457 belongs to the DUF464 family (57 members) for which there is no known function. The structure shows that it is composed of two helices in contact with one side of a five-stranded beta-sheet. Two identical monomers form a pseudo-dimer in the asymmetric unit. There is a large cleft between the first alpha-helix and the second beta-strand. This cleft may be functionally important, since the two highly conserved motifs, GHA and VCAXV(S/T), are located around the cleft. A structural comparison of TM1457 with known protein structures shows the best hit with another hypothetical protein, Ybl001C from Saccharomyces cerevisiae, though they share low structural similarity. Therefore, TM1457 still retains a unique topology and reveals a novel fold.

Amino Acid Motifs↗
PubMed2005

Crystal structures of an NAD kinase from Archaeoglobus fulgidus in complex with ATP, NAD, or NADP.

NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 A resolution, with its substrate NAD at 3.05 A resolution, and with the product NADP in two different crystal forms at 2.45 A and 2.0 A resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2' phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.

Adenosine Triphosphate↗
PubMed2005

Crystal structure of a heat-inducible transcriptional repressor HrcA from Thermotoga maritima: structural insight into DNA binding and dimerization.

All cells have a defense mechanism against a sudden heat-shock stress. Commonly, they express a set of proteins that protect cellular proteins from being denatured by heat. Among them, GroE and DnaK chaperones are representative defending systems, and their transcription is regulated by a heat-shock repressor protein HrcA. HrcA repressor controls the transcription of groE and dnaK operons by binding the palindromic CIRCE element, presumably as a dimer, and the activity of HrcA repressor is modulated by GroE chaperones. Here, we report the first crystal structure of a heat-inducible transcriptional repressor, HrcA, from Thermotoga maritima at 2.2A resolution. The Tm_HrcA protein crystallizes as a dimer. The monomer is composed of three domains: an N-terminal winged helix-turn-helix domain (WH), a GAF-like domain, and an inserted dimerizing domain (IDD). The IDD shows a unique structural fold with an anti-parallel beta-sheet composed of three beta-strands sided by four alpha-helices. The Tm_HrcA dimer structure is formed through hydrophobic contact between the IDDs and a limited contact that involves conserved residues between the GAF-like domains. In the overall dimer structure, the two WH domains are exposed, but the conformation of these two domains seems to be incompatible with DNA binding. We suggest that our structure may represent an inactive form of the HrcA repressor. Structural implication on how the inactive form of HrcA may be converted to the active form by GroEL binding to a conserved C-terminal sequence region of HrcA is discussed.

Bacterial Proteins↗
PubMed2005

Structure of a NAD kinase from Thermotoga maritima at 2.3 A resolution.

NAD kinase is the only known enzyme that catalyzes the formation of NADP, a coenzyme involved in most anabolic reactions and in the antioxidant defense system. Despite its importance, very little is known regarding the mechanism of catalysis and only recently have several NAD kinase structures been deposited in the PDB. Here, an independent investigation of the crystal structure of inorganic polyphosphate/ATP-NAD kinase, PPNK_THEMA, a protein from Thermotoga maritima, is reported at a resolution of 2.3 A. The crystal structure was solved using single-wavelength anomalous diffraction (SAD) data collected at the Se absorption-peak wavelength in a state in which no cofactors or substrates were bound. It revealed that the 258-amino-acid protein is folded into two distinct domains, similar to recently reported NAD kinases. The N-terminal alpha/beta-domain spans the first 100 amino acids and the last 30 amino acids of the polypeptide and has several topological matches in the PDB, whereas the other domain, which spans the middle 130 residues, adopts a unique beta-sandwich architecture and only appreciably matches the recently deposited PDB structures of NAD kinases.

Amino Acid Sequence↗
PubMed2005

Crystal structure of a nicotinate phosphoribosyltransferase from Thermoplasma acidophilum.

We have determined the crystal structure of nicotinate phosphoribosyltransferase from Themoplasma acidophilum (TaNAPRTase). The TaNAPRTase has three domains, an N-terminal domain, a central functional domain, and a unique C-terminal domain. The crystal structure revealed that the functional domain has a type II phosphoribosyltransferase fold that may be a common architecture for both nicotinic acid and quinolinic acid (QA) phosphoribosyltransferases (PRTase) despite low sequence similarity between them. Unlike QAPRTase, TaNAPRTase has a unique extra C-terminal domain containing a zinc knuckle-like motif containing 4 cysteines. The TaNAPRTase forms a trimer of dimers in the crystal. The active site pocket is formed at dimer interfaces. The complex structures with phosphoribosylpyrophosphate (PRPP) and nicotinate mononucleotide (NAMN) showed, surprisingly, that functional residues lining on the active site of TaNAPRTase are quite different from those of QAPRTase, although their substrates are quite similar to each other. The phosphate moiety of PRPP and NAMN is anchored to the phosphate-binding loops formed by backbone amides, as found in many alpha/beta barrel enzymes. The pyrophosphate moiety of PRPP is located at the entrance of the active site pocket, whereas the nicotinate moiety of NAMN is located deep inside. Interestingly, the nicotinate moiety of NAMN is intercalated between highly conserved aromatic residues Tyr(21) and Phe(138). Careful structural analyses combined with other NAPRTase sequence subfamilies reveal that TaNAPRTase represents a unique sequence subfamily of NAPRTase. The structures of TaNAPRTase also provide valuable insight for other sequence subfamilies such as pre-B cell colony-enhancing factor, known to have nicotinamide phosphoribosyltransferase activity.

Amino Acid Sequence↗
PubMed2005

Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications.

Type I restriction-modification enzymes are differentiated from type II and type III enzymes by their recognition of two specific dsDNA sequences separated by a given spacer and cleaving DNA randomly away from the recognition sites. They are oligomeric proteins formed by three subunits: a specificity subunit, a methylation subunit, and a restriction subunit. We solved the crystal structure of a specificity subunit from Methanococcus jannaschii at 2.4-A resolution. Two highly conserved regions (CRs) in the middle and at the C terminus form a coiled-coil of long antiparallel alpha-helices. Two target recognition domains form globular structures with almost identical topologies and two separate DNA binding clefts with a modeled DNA helix axis positioned across the CR helices. The structure suggests that the coiled-coil CRs act as a molecular ruler for the separation between two recognized DNA sequences. Furthermore, the relative orientation of the two DNA binding clefts suggests kinking of bound dsDNA and exposing of target adenines from the recognized DNA sequences.

Amino Acid Sequence↗
PubMed2005

Crystal structure of a PhoU protein homologue: a new class of metalloprotein containing multinuclear iron clusters.

PhoU proteins are known to play a role in the regulation of phosphate uptake. In Thermotoga maritima, two PhoU homologues have been identified bioinformatically. Here we report the crystal structure of one of the PhoU homologues at 2.0 A resolution. The structure of the PhoU protein homologue contains a highly symmetric new structural fold composed of two repeats of a three-helix bundle. The structure unexpectedly revealed a trinuclear and a tetranuclear iron cluster that were found to be bound on the surface. Each of the two multinuclear iron clusters is coordinated by a conserved E(D)XXXD motif pair. Our structure reveals a new class of metalloprotein containing multinuclear iron clusters. The possible functional implication based on the structure are discussed.

Amino Acid Motifs↗
PubMed2005

Global mapping of the protein structure space and application in structure-based inference of protein function.

We have constructed a map of the "protein structure space" by using the pairwise structural similarity scores calculated for all nonredundant protein structures determined experimentally. As expected, proteins with similar structures clustered together in the map and the overall distribution of structural classes of this map followed closely that of the map of the "protein fold space" we have reported previously. Consequently, proteins sharing similar molecular functions also were found to colocalize in the protein structure space map, pointing toward a previously undescribed scheme for structure-based functional inference for remote homologues based on the proximity in the map of the protein structure space. We found that this scheme consistently outperformed other predictions made by using either the raw scores or normalized Z-scores of pairwise DALI structure alignment.

Proteins↗