Analysis of structure and interactions of antimicrobial peptides in biological membrane as revealed by molecular dynamics simulation 分子動力学シミュレーション法を用いた生体膜中における抗菌ペプチドの構造と相互作用の解析

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著者

    • Javkhlantugs, Namsrai ジャブクラントゥクス, ナムスライ

書誌事項

タイトル

Analysis of structure and interactions of antimicrobial peptides in biological membrane as revealed by molecular dynamics simulation

タイトル別名

分子動力学シミュレーション法を用いた生体膜中における抗菌ペプチドの構造と相互作用の解析

著者名

Javkhlantugs, Namsrai

著者別名

ジャブクラントゥクス, ナムスライ

学位授与大学

横浜国立大学

取得学位

博士 (工学)

学位授与番号

甲第1441号

学位授与年月日

2012-03-23

注記・抄録

博士論文

本学位請求論文は、生体膜中における抗菌ペプチドの構造、配向及び相互作用について分子動力学シミュレーション法を用いて研究したものである。対象とした抗菌ペプチドは、ハチ毒のbombolitin II (BLT2)、および子牛の乳タンパクlactoferrinの構造中に含まれる抗菌活性部位と考えられているオリゴペプチドlactoferrampin(LFampinB)である。これらのペプチドは生体膜と相互作用して膜中に貫入し、生体膜中で特異な配向を示すことにより抗菌活性を示すと考えられている。本研究では,分子動力学シミュレーション法を用いた計算機化学的手法により、これらの抗菌ペプチドと生体膜との相互作用について詳細な研究を行ない、膜タンパク質であるBLT2 およびLFampinB の生体膜中における配向について、分子レベルでの相互作用機構を解明することが出来た。第1章 緒言 当該分野に関するこれまでの研究背景とその課題を示し、本研究の目的・意義について論じた。 第2章 分子動力学シミュレーション法の概説 本論文全体に渡って使用する分子動力学シミュレーション法について、その基礎と解析方法について概説した。 第3章 DPPC 2重膜中におけるbombolitin II (BLT2)の構造と相互作用 ハチ毒のbombolitin II (BLT2)が、dipalmitoylphosphatidylcholine (DPPC)脂質2重膜中で示す構造、配向状態を分子動力学シミュレーション法によって検討した。BLT2 の初期構造としてαへリックス構造を作成し、膜分子中に様々な位置、方向で配置して計算を行った。その結果、BLT2 はプラスの電荷を有するN 末端側が膜分子の表面近くに存在し、反対側の中性残基の多いC 末端側が膜中側に貫入した配置で、最も安定な状態を取り得ることが明らかになった。さらにBLT2 分子はαへリックス構造を安定に維持し、膜法線に対し傾いた角度で存在する様子もシミュレーションで再現することが出来た。そこで、分子間相互作用を詳細に検討した結果、BLT2 分子と膜分子の間に2か所の強い相互作用部位が見出された。ひとつは膜分子表面付近で、BLT2 分子のN 末端側と膜分子表面部位との強い相互作用。他方は、BLT2 分子のC 末端側と膜分子表面部位との間のクーロン静電相互作用である。BLT2 分子は膜表面付近の相互作用部位をアンカーとして、C 末端側がクーロン静電相互作用により膜表面方向に引き寄せられるため傾斜を示すことが明らかとなった。 第4章 DMPG 2重膜中における子牛Lactoferrampin (LFampinB)の構造と相互作用Lactoferrampin (LFampinB)はアミノ酸17残基からなる新規の抗菌ペプチドであり、子牛の乳タンパクであるlactoferrin の一部をなすオリゴペプチドである。酸性膜であるdimyristoylphosphatidylglycerol (DMPG)をモデル脂質膜として用い、その膜中での相互作用を分子動力学シミュレーション法により検討した。その結果、LFampinB は非解離型の末端を持つN 末端側の一部が膜分子の中に埋め込まれた構造となった。また、膜中部分はαへリックス構造を維持しており、そのヘリックス軸は膜の法線に対し、45°の角度をなして存在することも確認した。そこで、LFampinB と膜分子との分子間相互作用を詳細に検討し、構造の原因を分子レベルで明らかにした。 第5章 DMPC 2重膜中における子牛Lactoferrampin (LFampinB)の構造と相互作用 dimyristoylphosphatidylcholine (DMPC)は全電荷が0の中性膜であり、その膜分子とLFampinB との相互作用を分子動力学シミュレーションにより検討した。その結果、相互作用がDMPG の場合と比較して弱いことが示された。また、その相互作用の様子についてはDMPG との比較で、詳細な検討を行った。第6章 総括 膜中に存在するタンパク質の構造と機能の研究は分子生物学の中心課題の一つである。しかし、生体膜中で起こっている脂質分子とタンパク質の協同的なプロセスの解明は実験手段が限定されていることもありその結果も未だ不明な点が多い。本研究では、BLT2 やLFampinB などの膜タンパク質の脂質膜中における傾斜配向を分子動力学シミュレーション法により再現し、ペプチドに存在する特定の残基と膜分子とのクーロン静電相互作用がペプチドの膜中における配向に重要であることを明らかにした。このようなペプチドの膜中における配向は、今後膜タンパクが生体膜と相互作用し膜分子を破壊する機構を明らかにしていく際の基礎となることが期待される。

In present research work, we investigated the structure, orientation and interactions of peptides in membrane bilayer using molecular dynamics simulation. The antimicrobial peptides as bombolitin II (BLT2) and bovine lactoferrampin (LFampinB) are amphipathic peptides which are consists 17 amino acid residues with positively net charged. These peptides interact with lipid head groups of zwitterionic and negatively charged membrane bilayers which are the important factor of the tilting behavior of the peptides in the membrane. All the details are included in the thesis. The thesis consists 6 chapters and abstract of each chapter as follows. Chapter 1. Introduction This chapter is short introduction, where I briefly describe the basic biological active peptides and their biological activities of antibacterial and antimicrobial peptides. Objective of Present Work: We selected the antimicrobial peptides as BLT2 and LFampinB with positively net charged which are embedded peptide in the membrane. Chapter 2. Method of molecular dynamics simulation The background of the molecular dynamics (MD) simulation, the potential energy functions, the CHARMM force field and the analysis of the protein structure and function in CHARMM which were used that are presented in this chapter. Chapter 3. Bombolitin II (BLT2) in DPPC membrane bilayer Bombolitins are five structurally and functionally related heptadecapeptides originally isolated from venom of the bumblebee, Megabombus pennsylvanicus, and they lyse erythrocytes, release histamine from rat peritoneal mast cells, and enhance the activity of phospholipase A2. In our NMR experiment study, we performed solid?state 31P and 13C NMR experiment of Bombolitin II (BLT2) in a zwitterionic dipalmitoylphosphatidylcholine (DPPC) bilayer and revealed the dynamic structure and orientation of BLT2 peptide bound to DPPC membrane bilayer. It was revealed that the membrane?bound BLT2 adopts a straight ??helical structure and laterally diffuses in the membrane, rotating around the membrane normal with the tilt angle of the helical axis at 33?. We also performed the MD simulation of the BLT2 in DPPC membrane bilayer systems and showed the preliminary results that support our experimental results of the peptide structure and the orientation. The analysis of the structure, the interaction between the peptide and the membrane bilayer, and the mechanism of the orientation of the peptide in the membrane bilayer system were discussed in detail. BLT2 is an amphipathic peptide which consists of 17 amino acids with the sequence of Ser ? Lys ? Ile ? Thr ? Asp5 ? Ile ? Leu ? Ala ? Lys ? Leu10 ? Gly ? Lys ? Val ? Leu ? Ala15 ? His ? Val ? NH2. Ser1 at N-terminal was modeled with protonated form of NH3 + which is the dominant state at pH = 7. Val17 at C?terminal of was formed with the structure of CONH2 according to the amidation in our experimental procedures. This peptide has positive charges at the N?terminal of Ser1 and the residues of Lys2, Lys9, Lys12, and negative charge at the residue of Asp5 to give a net charge of +3. To build an initial conformation of BLT2, the lowest energy conformation of the peptide was first investigated because no crystal structure is available in the literature for this peptide. Energy minimization was performed using CHARMM with its all atom force field. The model of initial conformation of DPPC membrane bilayer used in this study was based on that developed by Feller et al. The peptide was placed in the DPPC Membrane bilayer, and most overlapped two DPPC lipid molecules, which have equivalent molecular weights with the peptide, were removed. MD simulation was performed using CHARMM with its all atom force field. The water models were represented using the modified TIP3P force field. The energy of the system was minimized by using two steps with crystal lattice. In the first step, the minimization was performed with the peptide main chain helical torsion angles constrained in the membrane. In the second step, the minimization was performed without any constraint on the peptide?membrane system. After the minimization, the MD simulation was performed without any constraint on the peptide?membrane system. After 20 ns MD simulation, we analyzed the distribution of the main chain torsion angles that shows no large deviation from the ??helical structure between residues from Lys2 to Ala15. The obtained tilt angle ??averaged for the last 5 ns is 51??to the membrane normal. Although this value is a little larger than the value of 33° obtained from the solid state NMR experiment, they are qualitatively in good agreement with each other and the tilting behavior of this peptide is clearly shown in this simulation. The strong interaction of Ser1 and Lys2 with the membrane head groups works as an “anchor” to keep these residues in this region during the tilting behavior. Lys9 and Lys12 are more important among all hydrophilic residues for rotational and translational movements of BLT2 in the membrane bilayer, because these hydrophilic residues locate far from the interface region. Lys9 interacts not only with lipid molecules but also with Asp5 to make an intra?molecular salt bridge. We showed the possible mechanism of the tilting behavior of the BLT2 molecule by using a MD simulation. The interaction exits between Lys12 and a lipid molecule through the hydrogen?bonding and electrostatic interaction. The understanding of the tilting behavior is important to elucidate the biological activity of this membrane peptide such as the signals and the toxics. Chapter 4. Bovine Lactoferrampin (LFampinB) in DMPG membrane bilayer Bovine lactoferrampin (LFampinB) is a new antimicrobial peptide which has 17 amino acids residues. This peptide was found in multifunctional glycoprotein, bovine lactoferrin and corresponding to 268?284 amino acid residues in the protein. LFampinB has stronger antimicrobial activity than parent protein and killed a wide variety of Gram?negative and Gram?positive bacteria such as E. coli and fungus as C. albicans. In this study, we revealed the local structure and interaction of LFampinB in the molecular level with acidic dimyristoylphosphatidylglycerol (DMPG) membrane. LFampinB is an antimicrobial peptide which consists of 17 amino acid residues as Trp ? Lys ? Leu ? Leu ? Ser5 ? Lys ? Ala ? Gln ? Glu ? Lys10 ? Phe ? Gly ? Lys ? Asn ? Lys15 ? Ser ? Arg. Trp1 at N?terminal was modeled with unprotonated form of ?NH2 and Arg17 at C?terminal was modeled with the structure of ?COO? same as experimentally synthesized procedures. This peptide has positively charges of the residues of Lys2, Lys6, Lys10, Lys13, Lys15, and negative charge at the residue of Glu5. The residue of Arg17 includes positively charged atoms. As a total, the peptide gives a net charge of +4. The initial conformation of LFampinB was built same as previous BLT2 peptide. In experimentally, the mixed membrane bilayers (mimetic bacterial membranes) with DMPG : DMPC : CL = 65 : 25 : 10 (w/w) were used. In MD simulation, DMPG (-1 charged) membrane with two 7 × 7 leaflets was used which were obtained by modified the palmitoyloleoylphosphatidylglycerol (POPG) membrane. As an initial orientation of the LFampinB in membrane, LFampinB was inserted into the membrane perpendicular to the membrane surface with its C?terminal close to the surface side by taking into account the NMR experimental data. MD simulation was performed for 10 ns. The MD results showed that LFampinB formed ??helical structure at N?terminal region and but residue 11 at the C-terminal side takes random coil conformation. The ??helix is tilted 42??in the membrane to the bilayer normal which is very close to the value of 45??obtained from solid?state NMR experiments. The rotation angle in helical wheel was 92.5??for Ile3 which is in excellent good agreement with solid?state NMR experimental value. We also analyzed the interaction between hydrophilic side chains and lipid head groups of membrane bilayer. The all detail was discussed in the thesis. Chapter 5. Bovine Lactoferrampin (LFampinB) in DMPC membrane bilayer In experimentally, the pure dimyristoylphosphatidylcholine (DMPC) (neutral) membrane were used and the ??helix is tilted 42??in the membrane. In MD simulation, DMPC membrane with two 7×7 leaflets was used which were obtained configuration of 8×8 leaflets of DMPC membrane. As an initial orientation of the LFampinB in membrane, LFampinB was inserted into the membrane perpendicular to the membrane surface with its C?terminal close to the surface side by taking into account the NMR experimental data. The MD simulation was performed for 30 ns. The MD results showed that LFampinB formed ??helical structure at N?terminal region 38??in the membrane to the bilayer normal which is very close to the value 42??obtained from solid?state NMR. We also analyzed the interaction between hydrophilic side chains and lipid head groups of membrane bilayer. The all detail was discussed in the thesis. Chapter 6. Conclusion of Thesis and Future works The structure and the function of the membrane proteins embedded in membrane are the central problems in molecular biology. However, the cooperative process of lipids and peptides occurring within the membrane is still very difficult to understand only by the experimental methods. Therefore, I studied molecular processes of antimicrobial peptides and lipids across the membrane by using MD simulation. Next, I will continue to study LFampinB in cardiolipin (CL) (-2 charged) membrane because CL is the key lipid to form pores in the bacterial membranes. I will elucidate the differences of the interaction between peptide and lipids to clarify each role of the membrane on the antibacterial mechanism. And, I especially want to investigate the orientation and interaction of the membrane with negatively and uncharged transmembrane (TM) peptides using solid?state NMR and MD simulation. The biological activities of these kinds of peptides are similar to the present research work although the sequence and the structure are different.

横浜国立大学, 平成24年3月23日, 博士(工学), 甲第1441号

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