PN01

Crystal Structures of the BlaI Repressor from Staphylococcus aureus and Its Complex with DNA: Insights into Regulation Mechanisms of

the bla and mec Operators

Tzu-Ping Ko*,1, Martin K. Safo2, Faik N. Musayev2, Howard Robinson3, Qixun Zhao2, Neel Scarsdale2,

Andrew H.-J. Wang1, and Gordon L. Archer2

1Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan; 2Virginia Commonwealth University, Richmond, VA 23298, USA; and 3Brookhaven National Laboratory, Upton, NY 11973, USA

 

BlaI is a 14 kDa protein that represses the transcription of blaZ, the gene encoding b-lactamase. It is homologous to MecI, which regulates the mecA gene of penicillin binding protein PBP2a. These genes mediate resistance to b-lactam antibiotics in Staphylococci. Both repressors can bind homologous and heterologous DNA promoter-operator sequences. The crystal structure of BlaI determined to 2.0 Å resolution shows that BlaI is a homodimer, similar to MecI, and each monomer contains a compact N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain that contains three a-helices. The dimeric BlaI is triangle shaped, in which the C-terminal domains are associated at the top vertex, and the individual N-terminal domains are on the base line. The crystal structure of BlaI in complex with the mec operator DNA determined to 2.7 Å resolution further shows conserved interactions between BlaI and DNA as observed in the MecI-DNA complex. Specific interactions between the recognition helix a3 of the protein and the conserved TACA/TGTA motifs of DNA are contributed mainly by the amino acid residues Thr44, Thr47, Arg51 and the nucleotides G and A of the TGTA motif. An interesting arrangement of BlaI dimers in an up-and-down manner along a virtual DNA double helix offers a possible explanation for cooperative and noncooperative binding of the repressors to the operators. Furthermore, the unbound BlaI dimer shows a closed conformation with the N-terminal domains rotated toward each other. Such flexibility may allow proteolytic inactivation of the repressor BlaI and MecI triggered by the penicillin receptors BlaR1 and MecR1.

 

 

PN02

Study of DNA triplex formation with two non-pyrimidine-purine-pyrimidine base triads in a Sept-decamer 5’-TTCTTCTGATTCTCTCC in Aqueous Solution

Min-Tasir Wey and Lou-Sing Kan

 

Institute of Chemistry, Academia Sinica, Taipei, Taiwan

 

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

 

Abstract : Triplex formation and stability of sept-decamer 5’-TTCTTCTGATTCTCTCC(C) with 5’-GGAGAGAATCAGAAGAA (W) and 5’-CCTCTCTTAGTCTTCTT (H) were studies by UV, CD, surface plasma resonance, and native gel electrophoresis as a function of pH. C, W, and H formed CWH triplex at low pH (5) in spite of two non-pyrimidine-purine-pyrimidine (pypupy) base triads in the middle of the oligomer.  This triplex dissociate when pH raising to neutral as a perfect pypupy triplex does. W and C but not H can self-associate into dimers also in an acidic environment by the results of native gel electrophoresis.  The W2 and C2 will dissociate by adding to each other to form CW.  In addition, WH forms by mixing W and H.   However, there is no CH dimer formed.  Thus, the relative affinity of C, H, and W components in triplex CWH were expressed thermodynamically and kinetically in this paper.  It is worth to note 5’-TTCTTCTGATTCCTCC is a fragment of cell cycle protein cdc25 gene (696-712) in pneomocystic carinii, a fungi that causes pneumonia  in patients with impaired immunity.  Thus, our study may aid the discovery of new drug designed against pneomocystic carinii.

 

  

PN03

DNA Binding and Cleavage by the Periplasmic Endonuclease Vvn from Vibrio vulnificus: A Novel Structure with a Known Active Site

Chia-Lung Li (李家隆)1,2, Lien-I Hor (何漣漪)3, Zee-Fen Chang (張智芬)2, Li-Chu Tsai (蔡麗珠)1, Wei-Zen Yang (楊維仁)1 and Hanna S. Yuan (袁小琀)1,2

1Institute of Molecular Biology, Academia Sinica, Nan-Kang, Taipei, Taiwan

2Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taiwan

3Department of Microbiology and Immunology, College of Medicine, National Cheng-Kung University, Tainan ,Taiwan

 

The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA. Vvn protects cells by preventing the uptake of foreign DNA during transformation. The crystal structure of the magnesium ion-bound Vvn and that of Vvn mutant H80A in complex with a duplex DNA and a calcium ion were resolved both at 2.3 Å resolution. Vvn has a novel mixed a/b topology containing four disulfide bridges. The overall structure of Vvn shows no similarity to other endonucleases, however, a known endonuclease motif containing a “bba-metal” fold is identified in the central cleft region. The crystal structure of the mutant Vvn/DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by about 20o. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting at structural basis for its sequence-independent recognition of DNA and RNA. Based on the enzyme/substrate and enzyme/product structures observed in the mutant Vvn/DNA crystals, a catalytic mechanism is proposed in which the His80 functions as a general base that activates a water molecule to attack the scissile phosphate, with a magnesium ion involved in stabilization of the phosphoanion transition state and in protonation of the 3’ oxygen. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA.

  

 

PN04

Heterodimeric Complexes of Hop2 and Mnd1 Function with Dmc1 to Promote Meiotic Homologous Juxtaposition and Strand-Assimilation

Yi-Kai Chen1,2*, Chih-Hsiang Leng1, Heidi Olivares4, Ming-Hui Lee1, Yuan-Chih Chang3, Wen-Mei Kung1, Shih-Chieh Ti1, Yu-Hui Lo1, Andrew H.-J. Wang1, Chia-Seng Chang3,
Douglas K. Bishop4, Yi-Ping Hsueh2, and Ting-Fang Wang1

1Institute of Biological Chemistry, 2Institute of Molecular Biology, and 3Institute of Physics,

Academia Sinica, Taipei, Taiwan and 4Department of Radiation and Cellular Oncology, University of Chicago, Illinois R.514, Int. of Biological Chemistry, Academia Sinica, No.128 Academia Road Section 2,

Nan-Kang, Taipei, 11529, Taiwan

 

Saccharomyces cerevisiae Hop2 and Mnd1 are abundant meiosis-specific chromosomal proteins, and mutations in the corresponding genes lead to defects in meiotic recombination and in homologous chromosome interactions during mid-prophase. Analysis of various double mutants suggests that HOP2, MND1, and DMC1 act in the same genetic pathway for the establishment of close juxtaposition between homologous meiotic chromosomes. Biochemical studies indicate that Hop2 and Mnd1 protein form a stable heterodimer with a higher affinity for double-stranded than single-stranded DNA, and that this heterodimer stimulates the strand assimilation activity of Dmc1 in vitro. Together, the genetic and biochemical results suggest that Hop2, Mnd1 and Dmc1 are functionally interdependent during meiotic DNA recombination.

 

 

PN05

NMR Evidence for Enzyme-Induced Strain in the Michaelis Complex of Serine Protease from SGNH-hydrolase Family with Paraoxon: Mechanism of Initial Noncovalent Binding.

 

Sergiy I. Tyukhtenko1*, Ching-Yu Chou1, Jei-Fu Shaw2 and Tai-huang Huang1

1Institute of Biomedical Sciences and 2Institute of Botany, Academia Sinica, Nan-Kang,

Taipei 11529, Taiwan, No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan

 

    Direct NMR observation of the fast formation of Michaelis complex (MC) of Thioesterase/Protease I (TEP-I) with paraoxon and its subsequent slow conversion to the transition state analogue complex (TSC) allowed us (Tyukhtenko et al. 2003, Biochemistry 42, 8289-8297) to reveal sequential stepwise structural changes of four conserved blocks comprising the active site along the reaction pathway. We focus here on the conformational changes of inhibitor on the formation of MC as well as TSC and we found that initial binding induced an unprecedented downfield 31P NMR chemical shift over 9 ppm relative to the free paraoxon while covalent binding induced only 0.2-0.8 ppm shift upfield from the signal of noncovalent complex. Analysis of 31P NMR data indicates that paraoxon undergoes a crucial conformational change only under initial noncovalent binding and conformation of paraoxon phosphate group is largely unaltered under covalent binding. Combined analysis of all heteronuclear NMR data for MC revealed that the active site in particular oxyanion hole of enzyme forces the inhibitor into the distorted conformation toward the transition state conformation. This is the first direct NMR evidence that the serine proteases use binding energy to strain or distort substrates in the MC. Structural and binding data suggest the dual role of the oxyanion hole in the SGNH-family: first in the strain/distortion of substrate ground state conformation toward transition state conformation in accompanying with weak hydrogen bonding and P=O bond polarization in the MC and then in subsequent stabilizing of tetrahedral transition state at the second step. The combination of structural, kinetic, mutagenesis and binding data allows us to conclude that stepwise structural rearrangements of TEP-I upon formation of TSC mostly represent sequential stepwise optimization of the oxyanion hole geometry of enzyme to adapt to each spice along the reaction coordinate. This work provide insight into chemical details and geometry optimization in the active site of serine proteases before chemical attack, as well as after covalent modification and can provide additional understanding for the ability of these enzymes to hydrolase the wide range of substrates. Supported by NSC Grant 91-2113-M-001-038 (R.O.C.)

 

 

PN06

Substrate and Product Specificities of Cis-type Undecaprenyl Pyrophosphate Synthase and Identification of General Base and Acid and the Role of

the Metal Ion during its Reaction

 

Annie P. C. Chen1*,  Sing-Yang Chang2,  Chih-Jung Kuo2,  Yang-Sheng Sun2,  Shih-Chun Li1 ,

Yu-Chung Lin3,  Chao-Tsen Chen3 ,  Andrew H.-J. Wang2,  and Po-Huang Liang1,2
1Institute of Biochemical Sciences and 3Department of Chemistry, National Taiwan University;

2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

No.128 Academia Road Section 2, Nankang, Taipei 11529, Taiwan

 

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP). UPP is a lipid carrier to mediate bacterial peptidoglycan synthesis. Our lab has been focusing on the multiple-step UPPs kinetics and defines its reaction mechanisms as well as its protein conformational changes. Furthermore, our three-dimensional structure of E.coli. UPPs has revealed an elongated hydrophobic tunnel-shaped crevice, and also demonstrated the role of a flexible loop. In this study, we would like to further investigate UPPs in case of substrate and product specificities, role of metal, and identification of general acid and base during catalysis. In terms of substrate binding, the pyrophosphate head group of FPP is bound with the positively-charged Arg residues and hydrocarbon moiety is bound with hydrophobic amino acids: Leu85, Leu88, and Phe89, located on a3 helix. Replacement of Leu85, Leu88, or Phe89 with Ala increase FPP and GGPP Km values by the same level, indicating that these amino acids are important for substrate binding but do not determine the substrate specificity. Furthermore, pyrophosphate of substrate is important since a synthetic FPP analogue with only one phosphate affect binding by nine folds. However, the length of hydrocarbone ate backbone, a five carbon longer of shorter substrate does not affect binding, and serves as an equally good substrate compared with FPP. Unlike trans-prenytransferases which all possess two DDxxD Asp-rich motifs to coordinate with two Mg2+ for binding with the pyrophosphates of the substrates, in UPPs which is cis-type, only one Mg2+ was found near IPP site but not directly chelate with pyroposphate. Substitution of Mg2+ with different metal ions led to different level of activity reduction. It was found that Mg2+  might not serve as a structure role in UPPs, rather, it was able to maintain the local structure stability near IPP. Moreover, we performed pH profile study of the UPPs reaction by measuring the kcat versus pH and in a novel way by examining the burst amplitude change with pH to derive the pKa of the essential general base His43. Another general base Asp26 was suspected to remove a proton from IPP.