Structure and function of peptide deformylase

Dissertation for Degree of Doctor Supervisor: Prof Lin-Woo Kang Structure and Function of Peptide Deformylase Submitted by HO THIEN HOANG February, 2018 Department of Biological Sciences Graduate School of Konkuk University Structure and Function of Peptide Deformylase A Dissertation submitted to the Department of Biological Sciences and the Graduate School of Konkuk University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Submitted by HO THIEN HOANG December, 2017 This certifies that the Dissertation of HO THIEN HOANG is approved Approved by Examination Committee: Chairman Member Member Member Member December, 2017 Graduate School of Konkuk University TABLE OF CONTENTS List of Tables…………………… …………………………………… iii List of Figures……… … …………………………………………… iv Abstract……….……………………………………………………… v Chapter Introduction 1.1 Peptide deformylase 1.1.1 The important of peptide deformylase in cell 1.1.2 Structure and mechanism of peptide deformylase 1.1.3 Xanthomonas oryzae pv oryzae 1.1.4 Acinetobacter baumannii 10 1.1.5 Three-dimensional structure determination methods 12 Chapter Methods 18 2.1 pv oryzae Cloning of peptide deformylase from Xanthomonas oryzae 18 2.2 Overexpression and purification of XoPDF 18 2.3 Crystallization, data collection, and structure determination of substrate- or inhibitor-bound XoPDF 21 2.4 Crystallization, data collection, and structure determination of fragment chemical compound-bound XoPDF 22 2.5 Structure determination and refinement of XoPDF 23 2.6 Cloning of AbPDF 24 2.7 Overexpression and purification of AbPDF 24 i 2.8 Crystallization and X-ray data collection of AbPDF 25 Chapter Results 27 3.1 Overall apo-XoPDF structure 27 3.2 Substrate-complex structure 36 3.3 Inhibitor-complex structure 39 3.4 Fragment chemical-complex structures 41 3.5 Overall apo-AbPDF structure 42 3.6 Inhibitor-complex AbPDF structure 47 Chapter Discussion 49 References 58 Abstract (in Korean) 66 ii List of Tables Table Data collection and refinement statistics 33 Table Data-collection and processing statistics 44 iii List of Figures Figure 1.1 Proposed catalytic mechanism of PDF Figure 1.2.Workflow for solving the protein structure by X-ray crystallography 13 Figure 1.3 Schematic illustration of a protein crystallization phase diagram 16 Figure 1.4 Two methods of preparing A: Hanging drop B: Sitting drop 17 Figure 2.1 Purified XoPDF is shown on a 15% SDS-PAGE gel 20 Figure 3.1 Native structure of XoPDF (a) 28 Figure 3.2 Metal binding and coordination of XoPDF 29 Figure 3.3 Sequence alignment of PDFs 30 Figure 3.4 Superimposed apo-, MA-bound, and MAS-bound XoPDF structures 31 Figure 3.5 Structures of PDF CD-loops 32 Figure 3.6 Refined electron density map of bound substrates and inhibitor actinonin 37 Figure 3.7 Substrate methionine-binding site 39 Figure 3.8 Comparison of actinonin-bound and FCC-bound XoPDF structures 40 Figure 3.9 Chemical structure and refined electron density maps of bound FCCs 42 Figure 3.10 Purified AbPDF on a 12% SDS-PAGE gel Molecular-mass markers are shown in lane M and purified AbPDF is shown in lane P 43 Figure 3.11 Native structure of AbPDF 46 Figure 3.12 Structure of CD-loop AbPDF 47 Figure 3.13 Superimposed apo-, Actinonin-bound AbPDF structure 48 Figure 4.1 Time-resolved transcriptional gene expression of XoPDF (Xoo1 075, def) and Xoo0585 genes 50 Figure 4.2 Sequence alignment of XoPDF and eukaryotic PDFs 52 Figure 4.3 Structure comparison between XoPDF and AtPDFs 53 Figure 4.4 Bottom view of substrate methionine-binding site 56 Figure 4.5 Exposure of Phe134 residue in XoPDF with the flexible open conformation of CD loop 57 Figure 4.6 Actinonin structure in XoPDF 57 iv ABSTRACT Structure and Function of Peptide Deformylase from Acinetobacter baumannii and Xanthomonas oryzae pv oryzae Ho, Thien Hoang Department of Biological Sciences Graduate School of Konkuk University Peptide deformylase (PDF) is an enzyme responsible for catalyzing the removal of the N-formyl group from the N-terminus following translation in bacterial cells and is an important target to develop antibacterial agents I determined crystal structures of two PDFs from Acinetobacter baumannii and Xanthomonas oryzae pv oryzae Acinetobacter baumannii is a Gramnegative opportunistic pathogen and has a high risk infection among immune impairment patients, particularly those who have stayed in hospital longterm Acinetobacter baumannii is one of the most common and serious multidrug-resistant pathogens Xanthomonas oryzae pv oryzae causes bacterial blight on rice; this species is one of the most devastating disease of rice worldwide In addition to the apo form of PDF structures, substrate and fragment chemical bound PDF structures were also determined, which include two substrate-bound (methionine-alanine or methionine-alaninev serine), an inhibitor-bound (actinonin), and six fragment chemical-bound structures Six fragment chemical compounds were bound in the substratebinding pocket The fragment chemical-bound structures were compared to the natural PDF inhibitor actinonin-bound structure The structural studies will be useful to design new inhibitors specific to AbPDF and XoPDF and potential antibiotics against Acinetobacter baumannii and Xanthomonas oryzae pv oryzae Key words: Acinetobacter baumannii, Xanthomonas oryzae pv oryzae, peptide deformylase, Bacterial blight (BB) disease, opportunistic bacterial pathogen vi Chapter Introduction 1.1 Peptide deformylase 1.1.1 The important of peptide deformylase in cell Peptide deformylase are metalloprotease in bacteria, plants (chloroplast), and humans (mitochondrial) that removes the formyl moiety on the methionine of Nformyl-methionine peptide in newly synthesized polypeptides which is essential process for them (Escobar-Alvarez et al., 2010) The protein synthesis usually stars with an N-formyl-methionine residue In its initiation complex, the key components in protein synthesis are the initiator fMet-tRNAfMet, three initiation protein factors (IF1, IF2, IF3), GTP, mRNA, a large 50S and a small 30S ribosomal subunits (Clark & Marcker, 1966; Laursen, Sorensen, Mortensen, & Sperling-Petersen, 2005; Marcker & Sanger, 1964) Initial there is a esterification of methionyl residue at the 3‟ end of initiator tRNAfMet, the methionine-tRNAfMet molecule is N-formylated by a methionyl-tRNAfMet formyltransformylase, so that the newly synthesized proteins are formylated at the amino terminus (Miesel, Greene, & Black, 2003) The formylation of the methionine tRNAfMet molecule is a crucial role in recognition of IF2 factor, and finally the codon is adjusted in the P site, contributing to the accuracy of translation initiation (Petersen, Roll, Grunberg-Manago, & Clark, 1979; Sundari, Stringer, Schulman, & Maitra, 1976) After the process of initiation, elongation of protein translation lead to the displacement of IF2 factor by EF-Tu molecules and GTP, which protect Met-tRNAfMet against enzymatic hydrolysis and between XoPDF and AtPDFs will provide useful information to develop a specific inhibitor against XoPDF Figure 4.3 Structure comparison between XoPDF and AtPDFs (a) Superimposed structures of actinonin-bound XoPDF (green) and MAS-bound AtPDF1A (brown) Actinonin in XoPDF is shown in yellow CD-loop of AtPDF1A is shaded in purple (b) Superimposed structures of actinonin-bound XoPDF (green) and actinonin-bound AtPDF1B (pale-blue) Actinonin in XoPDF is shown in yellow CD-loop of AtPDF1B is shaded in salmon (c) Superimposed substrate-binding site of actinonin-bound XoPDF (green) and MAS-bound AtPDF1A (brown) with the upper layer residues of the hydrophobic pocket for substrate methionine side chain (d) Superimposed substrate-binding site of actinonin-bound XoPDF (green) and 53 actinonin-bound AtPDF1B (pale-blue) with the upper layer residues of the hydrophobic pocket for substrate methionine side chain We determined crystal structures of XoPDF in complex with six FCCs in the substrate-binding pocket All FCCs were at least partially bound at the hydrophobic substrate methionine binding site, which could accommodate bulkier structural motifs such as rigid aromatic rings with conjugated π-bonds than the aliphatic side chain of methionine The ring structures were mainly stacked between one plane of His141 and Glu97 and another plane of Val45 (Figure 4.4) The binding of elongated ligand-like substrate MAS and actinonin in XoPDF caused opening of the CD loop, which revealed the concealed hydrophobic Phe134 in solution (Figure 4.5) The revealed Phe134 looked for a compensating hydrophobic moiety The terminal Five-member pyrrolidine ring of actinonin provided the compensating hydrophobic moiety and bound to Phe134 The Tyr69 residue in the CD loop had hydrophobic interactions with Phe134 in the closed conformation of the apo structure The angle between two aromatic rings of Phe134 and Tyr69 was approximately 42°, of which the partial and unparalleled interaction had an interaction of moderate strength and allowed plausible switching of the CD loop from closed to open conformation The PDF structure from Leptospira interrogans also showed the closed conformation of the CD loop in the apo structure(Zhou, Song, & Gong, 2005), and the tyrosine residue in the CD loop corresponding to XoPDF Tyr69 was conserved (Figures 3.2 and 3.3) Most other PDF structures exhibited an open CD loop conformation in the apo structure (Escobar-Alvarez et al., 2010; Moon, Park, & Kim, 2005; Smith et al., 2003) 54 (Figures 3.2) The position of Phe134 could also be an important site for the development of a specific inhibitor against XoPDF FCC5 and FCC6 exhibited different binding than other FCCs Both compounds bound from the methionine-binding site to the exposed Phe134 site as a direct line, which implies that the two binding sites are directly connected (Figures 3.8d) In the actinonin-bound XoPDF structure, the pentyl group and the other chain structure with the pyrrolidine ring of actinonin were ∼4 Å apart, which is a good distance for van der Waals interaction (Figures 4.6) The direct hydrophobic van der Waals interaction could hold actinonin as the same U-shape in water before binding to XoPDF, and the conformational rigidity of actinonin could increase the binding affinity for PDFs In summary, the hydrophobic substrate methionine-binding site is mainly involved in FCC binding via hydrophobic interaction and can accommodate bulky rigid aromatic ring structures The Phe134 residue provides an additional hydrophobic interaction site for ligands Crystal structures of XoPDF in complex with substrates and various FCCs may be useful for the development of new pesticides against Xoo 55 Figure 4.4 Bottom view of substrate methionine-binding site The fragment chemical compounds (FCCs)-bound structures were superimposed to the MA-bound structure and only FCCs and substrate methionine-binding site are shown in the same bottom view of Figure 3.7b 56 Figure 4.5 Exposure of Phe134 residue in XoPDF with the flexible open conformation of CD loop (a) The structure of closed CD loop (b) The structure of opened CD loop Figure 4.6 Actinonin structure in XoPDF The hydrophobic van der Waals interactions between the pentyl group and the other chain structure with the pyrrolidine ring are shown as black dashed lines The hydrophobic van der Waals interactions between Phe134 and the 5-member pyrrolidine ring is shown as blue arcs 57 References Adams, J M (1968) On the release of the formyl group from nascent protein J Mol Biol, 33(3), 571-589 Adams, J M., & Capecchi, M R (1966) N-formylmethionyl-sRNA as the initiator of protein synthesis Proc Natl Acad Sci U S A, 55(1), 147-155 Apfel, C M., Locher, H., Evers, S., Takacs, B., Hubschwerlen, C., Pirson, W., Keck, W (2001) Peptide deformylase as an antibacterial drug target: target validation and resistance development Antimicrob Agents Chemother, 45(4), 10581064 doi: 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X-ray diffraction data collected in oscillation mode Methods Enzymol, 276, 307-326 Zhang, H., & Wang, S (2013) Rice versus Xanthomonas oryzae pv oryzae: a unique pathosystem Curr Opin Plant Biol, 16(2), 188-195 doi: 10.1016/j.pbi.2013.02.008 Zhou, Z., Song, X., & Gong, W (2005) Novel conformational states of peptide deformylase from pathogenic bacterium Leptospira interrogans: implications for population shift J Biol Chem, 280(51), 42391-42396 doi: 10.1074/jbc.M506370200 65 ABSTRACT (in Korean) 펩타이드 디포밀라아제의 구조와 기능 연구 Ho, Thien-Hoang 생명과학과 건국대학교 대학원 Peptide deformylase (PDF)은 세균이 합성하는 새로운 polypeptide의 N말단에 존재하는 formyl group를 제거하는 역할을 하며, 이는 항균제 개발에 중요한 목표가 되는 효소로 여겨지고 있다 이번연구에서는 Acinetobacter baumannii 와 Xanthomonas oryzae pv Oryzae에 존재하는 2가지의 PDF들의 결정구조를 밝혔다 Acinetobacter baumannii는 가장 흔하고, 심각한 다중 약제 저항성 병원균이며, 그람음성의 기회주의적 병원균이며, 면역체계가 손상된 환자 중 특히, 병원에서 오랜 치료를 받은 환자에서 높은 감염률을 보여준다 세계의 쌀생산에서 가장 큰 파괴적 영향을 끼치는 병원체중 하나인 Xanthomonas oryzae pv Oryzae는 벼에 대하여 벼흰잎마른병을 일으킨다 이번연구에서는 PDF의 apo구조, 억제제, 기질, 화학물질 조각이 결합된 구조를 결정하였으며, 이에는 두가지 종류의 기질(methionine-alanine 이나 methionine-alanine-serine), 억제제(actinonin), 6가지의 화학물질 조각이 결합되는 구조가 있다 66 6가지의 화학물질 조각은 기질의 결합부위에 결합되었으며, 이 화학물질 조각들이 결합된 구조는 자연유래의 PDF 저해제인 actinonin이 결합된 구조와 비교할 수 있었다 이 구조적연구는 AbPDF와, XoPDF에 특이적인 새로운 억제제와, Acinetobacter baumannii 와 Xanthomonas oryzae pv Oryzae에 대한 항생제 개발에 용이하게 사용될 것이다 주제어: Acinetobacter baumannii, Xanthomonas oryzae pv oryzae, peptide deformylase, 세균성 마름병, 기회주의적 세균성 병원균 67 ... binding and coordination of XoPDF (a) Cd2+ ion in the metal site of MAS-bound XoPDF structure with 2Fofc and FoFc maps (b) Zn2+ ion in the metal site of MAS-bound XoPDF structure with 2Fofc and FoFc... Chapter Introduction 1.1 Peptide deformylase 1.1.1 The important of peptide deformylase in cell 1.1.2 Structure and mechanism of peptide deformylase 1.1.3 Xanthomonas oryzae.. .Structure and Function of Peptide Deformylase A Dissertation submitted to the Department of Biological Sciences and the Graduate School of Konkuk University in partial fulfillment of the