Honggao Yan

Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis.

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

Background: The methylerythritol phosphate (MEP) pathway is required for the biosynthesis of plastid-derived isoprenoids from plants. Results: Deoxyxylulose-5-phosphate synthase (DXS) was cloned from Populus trichocarpa, and metabolic regulation was tested. Conclusion: Both isopentenyl diphosphate and dimethylallyl diphosphate inhibit DXS by competing with thiamine pyrophosphate. Significance: Prediction of isoprene emission from trees and bioengineering of MEP pathway will be aided by these results. The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-d-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-d-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg2+ was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

Catalytic Center Assembly of Hppk as Revealed by the Crystal Structure of a Ternary Complex at 1.25 A Resolution

BACKGROUND Folates are essential for life. Unlike mammals, most microorganisms must synthesize folates de novo. 6-Hydroxymethyl-7, 8-dihydropterin pyrophosphokinase (HPPK) catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate pathway, and therefore is an ideal target for developing novel antimicrobial agents. HPPK from Escherichia coli is a 158-residue thermostable protein that provides a convenient model system for mechanistic studies. Crystal structures have been reported for HPPK without bound ligand, containing an HP analog, and complexed with an HP analog, two Mg(2+) ions, and ATP. RESULTS We present the 1.25 A crystal structure of HPPK in complex with HP, two Mg(2+) ions, and AMPCPP (an ATP analog that inhibits the enzymatic reaction). This structure demonstrates that the enzyme seals the active center where the reaction occurs. The comparison with unligated HPPK reveals dramatic conformational changes of three flexible loops and many sidechains. The coordination of Mg(2+) ions has been defined and the roles of 26 residues have been derived. CONCLUSIONS HPPK-HP-MgAMPCPP mimics most closely the natural ternary complex of HPPK and provides details of protein-substrate interactions. The coordination of the two Mg(2+) ions helps create the correct geometry for the one-step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transition state. The rigidity of the adenine-binding pocket and hydrogen bonds are responsible for adenosine specificity. The nonconserved residues that interact with the substrate might be responsible for the species-dependent properties of an isozyme.

Crystal structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, a potential target for the development of novel antimicrobial agents

BACKGROUND Folate cofactors are essential for life. Mammals derive folates from their diet, whereas most microorganisms must synthesize folates de novo. Enzymes of the folate pathway therefore provide ideal targets for the development of antimicrobial agents. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate biosynthetic pathway. RESULTS The crystal structure of HPPK from Escherichia coli has been determined at 1.5 A resolution with a crystallographic R factor of 0.182. The HPPK molecule has a novel three-layered alpha beta alpha fold that creates a valley approximately 26 A long, 10 A wide and 10 A deep. The active center of HPPK is located in the valley and the substrate-binding sites have been identified with the aid of NMR spectroscopy. The HP-binding site is located at one end of the valley, near Asn55, and is sandwiched between two aromatic sidechains. The ATP-binding site is located at the other end of the valley. The adenine base of ATP is positioned near Leu111 and the ribose and the triphosphate extend across and reach the vicinity of HP. CONCLUSIONS The HPPK structure provides a framework to elucidate structure/function relationships of the enzyme and to analyze mechanisms of pyrophosphoryl transfer. Furthermore, this work may prove useful in the structure-based design of new antimicrobial agents.

Kinetic and Thermodynamic Characterizations of Yeast Guanylate Kinase

Yeast guanylate kinase was expressed at high level in Escherichia coli using pET-17b vector. It was purified to homogeneity by a simple two-column procedure with an average yield of ∼100 mg/liter. The steady-state kinetic parameters for both forward and reverse reactions were determined by initial velocity measurements. The turnover numbers (kcat) were 394 s−1 for the forward reaction (formation of ADP and GDP) and 90 s−1 for the reverse reaction (formation of ATP and GMP). Km values were 0.20, 0.091, 0.017, and 0.097 mM for MgATP, GMP, MgADP, and GDP, respectively. Analysis of the initial velocity patterns indicated a sequential mechanism. GMP was found to have partial substrate inhibition. The substrate inhibition was not competitive with MgATP and could be attributed to formation of the abortive complex guanylate kinase·MgADP·GMP. The equilibrium constant of the reaction was measured under various conditions by NMR and a radiometric assay. The results showed that the steady-state kinetic parameters were consistent with the thermodynamic constant. NMR titration and equilibrium dialysis showed that both substrates and products could bind to free guanylate kinase. The dissociation constants were 0.090, 0.18, 0.029, 0.084, and 0.12 mM for MgATP, ATP, GMP, MgADP, and GDP, respectively. Viscosity-dependent kinetics was used to identify the rate-limiting steps of the reaction. The results indicated that the reaction rate is largely controlled by the chemical step.

Combinatorial Control of Human RNA Polymerase II (RNAP II) Pausing and Transcript Cleavage by Transcription Factor IIF, Hepatitis δ Antigen, and Stimulatory Factor II

When RNA polymerase II (RNAP II) is forced to stall, elongation complexes (ECs) are observed to leave the active pathway and enter a paused state. Initially, ECs equilibrate between active and paused conformations, but with stalls of a long duration, ECs backtrack and become sensitive to transcript cleavage, which is stimulated by the EC rescue factor stimulatory factor II (TFIIS/SII). In this work, the rates for equilibration between the active and pausing pathways were estimated in the absence of an elongation factor, in the presence of hepatitis δ antigen (HDAg), and in the presence of transcription factor IIF (TFIIF), with or without addition of SII. Rates of equilibration between the active and paused states are not very different in the presence or absence of elongation factors HDAg and TFIIF. SII facilitates escape from stalled ECs by stimulating RNAP II backtracking and transcript cleavage and by increasing rates into and out of the paused EC. TFIIF and SII cooperate to merge the pausing and active pathways, a combinatorial effect not observed with HDAg and SII. In the presence of HDAg and SII, pausing is observed without stimulation of transcript cleavage, indicating that the EC can pause without backtracking beyond the pre-translocated state.

Probing single-molecule enzyme active-site conformational state intermittent coherence.

The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Host target modification as a strategy to counter pathogen hijacking of the jasmonate hormone receptor

Significance Pathogen infections can cause significant crop losses worldwide and major disturbances in natural ecosystems. Understanding the molecular basis of plant disease susceptibility is important for the development of new strategies to prevent disease outbreaks. Recent studies have identified the plant jasmonate (JA) hormone receptor as one of the common targets of pathogen virulence factors. In this study, we modified the JA receptor and showed that transgenic Arabidopsis plants with the modified JA receptor gained resistance to bacterial pathogens that secrete a potent JA-mimicking toxin to promote infection. Our results suggest that host target modification may be developed as a new strategy to protect the disease-vulnerable components of the susceptible plant against highly evolved pathogens. In the past decade, characterization of the host targets of pathogen virulence factors took a center stage in the study of pathogenesis and disease susceptibility in plants and humans. However, the impressive knowledge of host targets has not been broadly exploited to inhibit pathogen infection. Here, we show that host target modification could be a promising new approach to “protect” the disease-vulnerable components of plants. In particular, recent studies have identified the plant hormone jasmonate (JA) receptor as one of the common targets of virulence factors from highly evolved biotrophic/hemibiotrophic pathogens. Strains of the bacterial pathogen Pseudomonas syringae, for example, produce proteinaceous effectors, as well as a JA-mimicking toxin, coronatine (COR), to activate JA signaling as a mechanism to promote disease susceptibility. Guided by the crystal structure of the JA receptor and evolutionary clues, we succeeded in modifying the JA receptor to allow for sufficient endogenous JA signaling but greatly reduced sensitivity to COR. Transgenic Arabidopsis expressing this modified receptor not only are fertile and maintain a high level of insect defense, but also gain the ability to resist COR-producing pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. Our results provide a proof-of-concept demonstration that host target modification can be a promising new approach to prevent the virulence action of highly evolved pathogens.

NMR detection of bifurcated hydrogen bonds in large proteins.

Hydrogen bonds play critical roles in protein structure, stability, and function. Conventionally, hydrogen bonds are mainly determined by X-ray crystallography and NOE-based NMR spectroscopy in indirect manners. In recent years, it was demonstrated that hydrogen bonds can be directly detected through NMR measurements of trans-hydrogen-bond scalar coupling constants. Here we report across hydrogen-bond protium/deuterium isotope effects in a 35 kDA protein observed with the isotopomer-selective TROSY NMR technique (Liu et al. J. Biomol. NMR 2006, 36, 205−214; Liu et al. J. Magn. Reson. 2007, 186, 319−326) and show that such isotope effects can be used to detect a most common type of bifurcated hydrogen bonds, in which a heavy atom, usually oxygen, is involved in two hydrogen bonds, including a pair of bifurcated hydrogen bonds involving a bound water molecule.

Structure-function relationships of cellular retinoic acid-binding proteins. Quantitative analysis of the ligand binding properties of the wild-type proteins and site-directed mutants.

It has been suggested that electrostatic interactions are critical for binding of retinoic acid by cellular retinoic acid-binding proteins (CRABP-I and CRABP-II). However, the roles of two conserved arginine residues (Arg-111 and Arg-131 in CRABP-I; Arg-111 and Arg-132 in CRABP-II) that interact with the carboxyl group of retinoic acid have not been evaluated. A novel competitive binding assay has been developed for measuring the relative dissociation constants of the site-directed mutants of CRABPs. Arg-111 and Arg-132 of CRABP-II were replaced with methionine by site-directed mutagenesis. The relative dissociation constants of R111M and R132M (Kd (R111M)/Kd (CRABP-II) and Kd (R132M)/Kd(CRABP-II)) were determined to be 40-45 and 6-8, respectively. The ring protons of the aromatic residues of the wild-type CRABP-II and the two mutants were sequentially assigned by two-dimensional homonuclear NMR in conjunction with three-dimensional heteronuclear NMR. Detailed analysis of the nuclear Overhauser effect spectroscopy spectra of the proteins indicated that the conformations of the two mutants are highly similar to that of the wild-type CRABP-II. These results taken together showed that Arg-111 and Arg-132 are important for binding retinoic acid but contribute to the binding energy only by ∼2.2 and 1.2 kcal/mol, respectively. In addition, the relative dissociation constant of CRABP-II and CRABP-I (Kd (CRABP-II)/Kd (CRABP-I)) was determined to be 2-3, in close agreement with that calculated using the apparent Kd values determined under the same conditions by fluorometric titrations.