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Structural Characterization and Subunit Communication Of STRUCTURAL CHARACTERIZATION AND SUBUNIT COMMUNICATION OF ESCHERICHIA COLI PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX By Jaeyoung Song A dissertation submitted to the Graduate School-Newark Rutgers, The State University of New Jersey In partial fulfillment of requirements for the degree of Doctor of Philosophy Graduate Program in Chemistry Written under the direction of Professor Frank Jordan and approved by Newark, New Jersey May, 2011 ABSTRACT OF THE THESIS Structural Characterization and Subunit Communication of Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex By Jaeyoung Song Thesis Director: Professor Frank Jordan The pyruvate dehydrogenase multienzyme complex (PDHc) from Escherichia coli (E. coli) is the best characterized of the 2-oxoacid dehydrogenase complexes. The complex plays a role as catalyst for the conversion of pyruvate to acetyl Coenzyme A (acetylCoA) by three enzyme components in the complex. The complex is comprised of 24 copies of the dimeric pyruvate dehydrogenase (E1ec; 99,474 Da), a cubic core of 24 copies of dihydrolipoamide acetyltransferase (E2ec; 65,959 Da), and 12 copies of dihydrolipoamide dehydrogenase (E3ec; 50,554 Da) (1-3). The crystal structure of the E. coli pyruvate dehydrogenase complex E1 subunit (E1ec) has been deterimined, and there were three missing regions (residues 1-55, 401-413, and 541-557) remaining absent in the model due to high flexibilities of these regions (4). Most bacterial pyruvate dehydrogenase complexes from either Gram-positive or Gram-negative bacteria have E1 components with an 2 homodimeric quaternary structure. In a sequel to our previous publications (5-8), the first NMR study on the flexible regions of the E1 component from Escherichia coli and its biological relevance ii was presented. In the study, sequence-specific NMR assignments for six residues in the N-terminal 1-55 region, and for a glycine in each of the two mobile active center loops of the E1 component, a 200 kDa homodimer was made. This was accomplished by using site-specific substitutions and appropriate labeling patterns, along with a peptide with the sequence corresponding to the N-terminal 1-35 amino acids of the E1 component. To study the functions of these mobile regions, the spectra were also examined in the presence of: (a) a reaction intermediate analog known to affect the mobility of the active center loops, (b) an E2 component construct consisting of a lipoyl domain (LD) and peripheral subunit binding domain (PSBD) and (c) a peptide corresponding to the amino acid sequence of the E2 peripheral subunit binding domain. Deductions from the NMR studies are in excellent agreement with our functional finding, providing clear indication that the N-terminal region of the E1 interacts with the E2 peripheral subunit binding domain, and that this interaction precedes reductive acetylation. The results provide the first structural support to the notion that the N-terminal region of the E1 component of this entire class of bacterial pyruvate dehydrogenase complexes is responsible for binding the E2 component. Among three components of PDHc, E2ec consists of 24 chains, and in the overall reaction, the lipoyl domain is reductively acetylated by E1ec and pyruvate, and S- acetyldihydrolipoyl domain transfers acetyl group to Coenzyme A leading acetylcoenzyme A production. Even though the precise number of E2 subunits is still ambiguous, in many cases including human PHDc (9), the sum is a multiple of three chains indicating that multiples of chains can affect the acetyl transfer to CoA by interchain acetyl transfer. To answer this question, the E2 component from E. coli iii specifically designed with only a single lipoyl domain (LD, 1-lip E2ec), rather than the three lipoyl domains found in the wild type enzyme (3-lip E2ec) was used. Earlier, it was shown that the activities of these two enzymes are virtually the same, while the 1-lip E2ec provides obvious advantages for mechanistic studies (10). At the same time, it is also important to point out that there indeed are other sources of the E2 component with only a single domain, such as from Mycobacterium tuberculosis. To study the question, two constructs of the 1-lip E2ec were prepared, one in which the lysine on the LD ordinarily carrying the lipoic acid is changed to alanine (henceforth K41A), and a second one in which the histidine believed to catalyze the transacetylation in the catalytic domain (CD) is substituted to A or C, H399C and H399A. The first is incompetent towards posttranslational ligation of the lipoic acid, hence towards reductive acetylation. The second one is incompetent towards acetylCoA formation, by virtue of the absence of the catalytic histidine residue. This is a biochemical version of a classical crossover experiment, as should the reaction proceed within one chain the two constructs should each be inactive either together or individually. On the other hand, should the reaction proceed by an interchain mechanism, addition of the two constructs should produce measurable activity. Both kinetic and mass spectrometric evidence supported the second scenario. Hence, plausible model/explanation for the multiples of three chains present in each E2 component as well as for their assembly was suggested. iv ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my advisor Prof. Frank Jordan for the continuous support, motivation, enthusiasm, and immense knowledge of my Ph.D study. I would like to thank to Dr. Natalia Nemeria for the help and the contribution of didomain E2ec, thank to Dr. Yun-Hee Park for the contribution of human didomain and tridomain, and thank to Dr. Sachin Kale for the contribution of His6-tag E1ec. Last but not least, I would like to thank to my thesis committees, Prof. Edgardo T. Farinas, Prof. Richard Mendelsohn, and Prof. Darren Hansen for kind consideration of my thesis, to my family for the immense support, to Dr. Lazaros Kakalis for the help with NMR, to Dr. Roman Brukh for the help with FTMS, and to Anand Balakrishnan for sharing of research interests and discussion. v TABLE OF CONTENTS ABSTRACT OF THE THESIS .......................................................................................... ii ACKNOWLEDGEMENTS ................................................................................................ v LIST OF TABLES ............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii LIST OF SCHEMES........................................................................................................ xvi ABBREVIATIONS ........................................................................................................ xvii CHAPTER 1. Structural and Functional Characterization of N-Terminal Region in the E1ec Component of the Pyruvate Dehydrogenase Complex .............................................. 1 1.1. INTRODUCTION ................................................................................................. 1 1.2. MATERIALS and METHODS ............................................................................. 6 1.2.1. Materials ......................................................................................................... 6 1.2.2. Plasmid purification ........................................................................................ 7 1.2.3. Site directed mutagenesis ................................................................................ 9 1.2.4. Protein expression ......................................................................................... 14 1.2.5. Protein purification ....................................................................................... 16 1.2.6. 1-lip E2ec purification .................................................................................. 18 1.2.7. His6-tag didomain of 1-lip E2ec (E21-190) purification ................................. 20 1.2.8. Purification of His6-tag lipoyl domain of 1-lip E2ec .................................... 22 1.2.9. E3ec purification ........................................................................................... 23 vi 1.2.10. SDS-PAGE ................................................................................................ 24 1.2.11. Concentrating protein using Millipore Centriprep Centrifugal Filter Units 26 1.2.12. Determination of protein concentration by Bradford assay ....................... 27 1.2.13. Overall activity measurement .................................................................... 29 1.2.14. Structural analysis of mobile regions of E1ec using Nuclear Magnetic Resonance .................................................................................................................. 29 1.3. RESULTS ............................................................................................................ 31 1.3.1. Kinetic properties of the C-terminal His6-tag E1ec indicates an acceptable surrogate for the non His6-tag version ....................................................................... 31 1.3.2. 1H-15N TROSY-HSQC spectrum shows well dispersed resonances, but only a limited number from E1ec ...................................................................................... 34 1.3.3. Glycine auxotroph assisted in the assignment of resonances to glycines ..... 36 1.3.4. 3D HNCO revealed Glycine 47 in N-terminal region
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