NMR-Based Computational Studies of Membrane Proteins in Explicit Membranes

Since nuclear magnetic resonance (NMR) spectroscopy data, including solution NMR from micelles and solid-state NMR from bilayers, provide valuable structural and dynamics information of membrane proteins, they are commonly used as restraints in structural determination methods for membrane proteins. However, most of these methods determine the protein structures by fitting the single-confer model into all available NMR restraints regardless of the explicit environmental effects that are determinant in the structures of membrane proteins. To develop a reliable protocol for obtaining optimal structures of membrane proteins in their native-like environments, various NMR properties were applied in the refinement approaches using explicit molecular dynamics (MD) simulations in this research. First, solution NMR NOE based-distance measurements were used as restraints in MD simulations to refine an activating immunoreceptor complex in explicit environments. Compared to the structure determined in vacuum, the resulting structures from the explicit restrained simulations yields a more favorable and realistic side-chain arrangement of a key Asp residue, which is highly consistent with mutagenesis studies on such residue. Incorporating solid-state NMR and solution NMR, MD simulations were performed in the explicit bilayers to refine the structure of membrane-bound Pf1 coat protein. Since solid-state NMR is sparse in its N-terminal periplasmic helix, the protein structure was determined by combining solid-state NMR and solution NMR. Benefiting from the sophisticated energy function and the explicit environments in MD, the orientation of Pf1's periplasmic helix can be identified in simulations restrained by solid-state NMR alone. In the simulations restrained with both solid-state NMR and solution NMR, physically irrelevant structures were frequently observed, suggesting there are conflicts between the restraints from different sample types (e.g., bilayers and micelles). As NMR data are ensemble-averaged measures, the solid-state NMR restrained explicit ensemble dynamics (ED) simulations of fd coat protein were performed in different ensemble sizes and compared to the unrestrained MD simulations. As the ensemble size increases, the violations of resulting structures from experimental NMR data decrease, while the structural variations increase to be comparable to the unrestrained MD simulations, indicating the efficacy of restrained ED in refining structures and extracting dynamics. To investigate the influence of different environments on the structures of membrane proteins, in this research, MD simulations were performed in bilayers and micelles, respectively. Since building a preassembled protein/micelle complex for MD simulation is challenging and requires considerable experience with simulation software, a web-based graphical interface Micelle Builder in CHARMM-GUI (http://www.charmm-gui.org/input/micelle) was developed to support users to build micelle systems in a automatic and simplified process. Using this interface, Pf1 coat protein was preassembled in a protein/micelle model and simulated in explicit environment. Compared to previous simulations of Pf1 coat protein in bilayers, different protein conformations were observed in these simulations due to the distinct behavior and geometry of micelles

CHARMM-GUI Micelle Builder for Pure/Mixed Micelle and Protein/Micelle Complex Systems

<i>Micelle Builder</i> in CHARMM-GUI, http://www.charmm-gui.org/input/micelle, is a web-based graphical user interface to build pure/mixed micelle and protein/micelle complex systems for molecular dynamics (MD) simulation. The robustness of <i>Micelle Builder</i> is tested by simulating four detergent-only homogeneous micelles of DHPC (dihexanoylphosphatidylcholine), DPC (dodecylphosphocholine), TPC (tetradecylphosphocholine), and SDS (sodium dodecyl sulfate) and comparing the calculated micelle properties with experiments and previous simulations. As a representative protein/micelle model, Pf1 coat protein is modeled and simulated in DHPC micelles with three different numbers of DHPC molecules. While the number of DHPC molecules in direct contact with Pf1 protein converges during the simulation, distinct behavior and geometry of micelles lead to different protein conformations in comparison to that in bilayers. It is our hope that CHARMM-GUI <i>Micelle Builder</i> can be used for simulation studies of various protein/micelle systems to better understand the protein structure and dynamics in micelles as well as distribution of detergents and their dynamics around proteins

돌연변이와 인산화에 의한 핵 라민의 조립과정에 대한 구조적 연구

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2023. 2. 하남출.세포핵 중간세사 라민은 핵의 형태를 유지하고 외부의 기계적 자극으로부터 대항하는 힘을 갖는 3차원 그물망 구조를 형성한다. 핵 라민은 세포의 생명유지에 중요하다. 라민은 유전자 돌연변이에 의해 다양한 laminopathies가 나타나는데, 그 중 S143F의 유전자 돌연변이는 프로게리아와 근육 파괴를 모두 특징으로 하는 표현형을 유발한다. 본 연구에서는 S143F변이를 가진 라민 A/C의 단백질 결정 구조를 규명하였다. 라민 A/C S143F의 구조에서 페닐알라닌으로 치환된 143번 잔기는 소수성 상호작용에 의해 결정구조에서 사량체의 코일들 사이의 X 자형 상호작용을 보여주었다. 후속연구는 필라멘트 사이의 X자형 상호작용이 정상적인 라민 그물망 구조를 방해하는데 중요한 역할을 한다는 것을 보여주었다. 이 연구 결과는 3차원 그물 구조의 조립 메커니즘을 제안하고 핵 변형에 의해 노화과정을 이해하기 위한 분자적 수준의 토대를 추가로 제공한다. 핵 라민은 A11과 A22 결합 방법으로 알려진 코일형 이량체의 두가지 분자 배열을 통해 길고 선형의 필라멘트를 형성함으로써 핵의 구조를 유지한다. 라민의 사량체 형성과정에서 A11과 A22와 결합하는 코일형 이중체 사이의 결합은 ACN 결합이라는 또다른 평행한 코일 1a와 코일 2의 카르복실 말단 사이의 머리-꼬리 상호작용을 형성한다. 체세포분열 동안 CDK1 인산화효소 복합체에 의해 라민의 아미노 말단의 머리 부분의 인산화는 핵 라민의 해체를 유발하지만, 이에 관한 분자수준의 메커니즘은 알려지지 않은 상태로 남아있다. 본 연구에서는 정제된 단백질을 이용하여 CDK1 복합체에 의한 인산화는 코일 1a와 코일 2의 C-말단 사이의 ACN 결합을 직접적으로 방해함으로써 섬유질 라민의 분해를 촉진한다는 것을 밝혔다. 또한 코일 1a와 코일 2 사이의 결합이 아미노산 잔기간의 이온 결합력의 변화로 인해 중단되었음을 관찰하였습니다. 덧붙여 분자모델을 곁들여 라민이 CDK1에 의존한 해체 메커니즘을 제시하였다. L59R 돌연변이에 의한 laminopathy는 근육파괴질환에서 골격근육질병과 심장근육병증의 표현형을 모두 유발한다. 이 유전자 돌연변이는 coil 1a의 안정도를 변화시켜 ACN 결합을 강하게 유도하며, 이는 인산화효소에 의한 라민의 해체 시도를 저지할 정도로 강력하다는 것을 앞서 실험을 통해 확인하였다. 본 연구에서는 이 결합을 억제하는 천연물을 스크리닝 하였고 플라보노이드 계열의 모린, 바이칼레인, 피세틴, 그리고 아피제닌을 선정하였다. 분자 도킹과 분자 동력학 시뮬레이션을 활용하여 이들의 분자적 작동기전에 대한 단서를 제공하였다. 그리고 HT1080 세포 기반 분석은 라민 A의 비정상적 상호작용에 플라보노이드가 효과적인 분자로서 활용 가능성을 보여주었다. 이러한 결과들은 라민의 유전질병에서 플라보노이드가 항산화 기능 외에 단백질의 결합방해에 직접적으로 관여하여 치료제로서의 활용 가능성을 보여준다.Intermediate filament lamins in the cell nucleus form a three-dimensional meshwork that maintains the shape of the nucleus and offers a framework against external mechanical stress. The nuclear lamin is crucial to maintain the life of a cell. Lamin has various laminopathies caused by genetic mutations, of which the gene mutation of S143F has caused phenotypes characterized by both progeria and muscular dystrophy. This study determined the crystal structure of the lamin A/C fragment harboring the S143F mutant. The obtained structure revealed an X-shaped interaction between the tetrameric units in the crystals, potentiated by the hydrophobic interactions of the mutated Phe143 residues. Subsequent studies indicated that the X-shaped interaction between the filaments is crucial for disrupting the normal lamin meshwork. These results suggest the assembly mechanism of the 3-D meshwork and provide a molecular framework for understanding the aging process by nuclear deformation. Nuclear lamins maintain the nuclear envelope structure by forming long, linear filaments via two alternating molecular arrangements of coiled-coil dimers, known as A11 and A22 binding modes. The coupling between the coiled-coil dimer that binds to A11 and A22 during the lamin tetramer formation produces another parallel head-to-tail interaction between coil 1a and the C-terminal region of coil 2, called the ACN interaction. During mitosis, phosphorylation in the lamin N-terminal head region by the cyclin-dependent kinase (CDK) complex triggers the depolymerization of lamin filaments, but the associated molecular-level mechanisms remain unknown. This study revealed that phosphorylation by the CDK1 complex promotes the disassembly of lamin filaments by directly interfering with the ACN interaction between coil 1a and the C-terminal portion of coil 2 using purified proteins. Furthermore, it was observed that this interaction was disrupted as a result of alteration in the ionic interactions between coil 1a and coil 2. In addition, the disassembly mechanism of CDK1-dependent lamin filaments was presented in combination with molecular modeling. The L59R mutation of lamin-induced laminopathy causes cardiomyopathy and Malouf syndrome phenotypes in muscular dystrophy. It was confirmed through previous experiments that this gene mutation changes the stability of coil 1a to strongly induce ACN interaction, which is strong enough to restrain attempts to disperse lamin by kinase. This research screened a natural compound that inhibits ACN interaction and selected the flavonoids morin, baicalein, fisetin, and apigenin. Molecular docking and molecular dynamics simulations were used to explain their molecular mechanisms. Furthermore, HT1080 cell-based assays showed the possibility of using flavonoids as effective molecules for abnormal interactions of lamin A. These results show the possibility that flavonoids are directly involved in both the inhibition of protein binding and antioxidant function in the genetic diseases of lamin and can be used as a therapeutic agent.Chapter 1. Background 1 1.1. The role of nuclear lamins in cells ２ 1.1.1. Expression of nuclear lamins and gene organization ２ 1.1.2. Characteristic structural features of lamin ３ 1.1.3. Lamins regulate nuclear mechanics ４ 1.1.4. Lamins regulate chromatin organization and DNA damage and repair ５ 1.2. Classification of laminopathies ７ 1.2.1. Muscular dystrophy ７ 1.2.2. Lipodystrophy ７ 1.2.3. Hutchinson Gilford Progeria Syndrome (HGPS) ８ 1.3. Flavonoid ９ 1.3.1. Classification of flavonoid ９ 1.3.2. Flavonoid as an anti-aging agent ９ 1.4. Purpose of Research 10 Chapter 2. Crystal structure of progeria mutant S143F lamin A/C and its implications for premature aging 11 2.1. Introduction 12 2.2. Materials and Methods 15 2.2.1. Plasmid construction 15 2.2.2. Purification of the recombinant proteins 15 2.2.3. Crystallization, structure determination, and analysis 16 2.2.4. Pull-down assays 19 2.3. Results 20 2.3.1. The overall structure of the lamin S143F mutant protein 20 2.3.2. The S143F mutation does not change the interactions to make the linear filament 24 2.3.3. Phe143-mediated x-shaped interaction between the A11 tetramers ２８ 2.3.4. Synergistic aggregation of the S149C mutation near Phe143 in the cell ３３ 2.4. Discussion ３６ Chapter 3. Cyclin-dependent Kinase 1 depolymerizes nuclear lamin filaments by disrupting the head-to-tail interaction of the lamin central rod domain. 40 3.1. Introduction 41 3.2. Materials and Methods 46 3.2.1. Expression and purification of proteins 46 3.2.2. Phosphorylation of the lamin N-terminal fragment 47 3.2.3. GST pull-down assays 47 3.2.4. Lamin coil 2-conjugated Sepharose pull-down assay 48 3.2.5. Circular dichroism (CD) 48 3.2.6. Lamin complex modeling 49 3.3. Results 50 3.3.1. The phosphorylation of lamin by CDK1 inhibits the A22 interaction 50 3.3.2. Phosphorylation of Thr19 and Ser22 inhibits the ACN mode 56 3.3.3. The phosphorylation of lamin A/C/ does not affect the coiled-coil structure of coil 1a 59 3.3.4. Synergistic effects of the other cellular kinases with CDK1 activity on lamin 65 3.3.5. The importance of the ionic interaction in the ACN binding 67 3.4. Discussion 70 Chapter 4. The flavonoid alleviates incorrect lamin assemblies by interrupting the ACN interaction of lamin A 75 4.1. Introduction 76 4.2. Materials and Methods 78 4.2.1. Protein expression and purification 78 4.2.2. ELISA 78 4.2.3. Molecular docking and molecular dynamics simulation studies 79 4.2.4. Human fibrosarcoma cell culture and flavonoid treatment 79 4.3. Results 83 4.3.1. Screening of inhibitory compound for abnormal ACN interactions in nuclear lamin L59R mutant 83 4.3.2. Predicting the effect of flavonoids on abnormal interaction of lamin A 87 4.3.3. The flavonoid improves the nuclear deformation formed by an abnormal interaction 93 4.4. Discussion 96 Bibliography 98 국문초록 110박

Computational Molecular Biophysics of Membrane Reactions

Proteins are nanoscale molecules that perform functions essential for biological life. Membranes surrounding cells, for example, contain receptor proteins that mediate communication between the cell and the external milieu, membrane transporters that transport ions and larger compounds across the membranes, and enzymes that catalyze chemical reactions. Likewise, soluble proteins found in interior of the cell include motor proteins that move other proteins around, enzymes that bind to and repair breaks in the DNA, and proteins that help control the cellular clock. Mutations in genes that encode proteins can cause disease, as is the case of cystic fibrosis, a disease that associates with mutation of a chloride channel called the cystic fibrosis transmembrane conductance regulator.1 The essential functions they perform in the cell makes proteins essential drug targets for modern bio-medical applications. An important example here is the programmed death ligand-1 (PD-L1), which is a valuable target for modern immunotherapy.2-4 Predicting how a protein responds to a drug molecule, or using the protein as inspiration for biotechnological applications, require knowledge of how that protein works. As proteins are dynamic entities and protein dynamics are essential for function,5-8 describing the mechanism of action of a protein requires knowledge about the protein motions in fluid environments. Theoretical biophysics provides valuable tools to characterize protein reaction mechanisms and protein motions at the atomic level of detail. This Habilitation Thesis presents research on using theoretical biophysics approaches to decipher how proteins work. The focus of the research is on membrane proteins and reactions that occur at lipid membrane interfaces. The central question I address is the role of dynamic hydrogen (H) bonds in protein function and membrane interactions. The methods used include quantum mechanical (QM) computations of small molecules, combined quantum mechanics/molecular mechanics (QM/MM) of chemical reactions in protein environments, classical mechanical computations of large protein and membrane systems, and bridging numerical simulations to bioinformatics. In my research group we developed algorithms to identify H-bond networks in proteins and membrane environments, and to characterize the dynamics of these networks. To extend the applicability of numerical computations to bio-systems that bind drug-like compounds, we derive parameters for a potential energy function widely used in the field. The main research topics and specific questions addressed are summarized below together with a discussion of the computational approaches used

Interpretation of in-solution small-angle scattering data using molecular dynamics simulations

The accurate determination of macromolecular structures often necessitates joint experimental and computational efforts. In this thesis, MD simulations are engaged to interpret small-angle scattering data of X-rays (SAXS) and neutrons (SANS). SAXS and SANS experiments are performed under near-native conditions, but provide only limited amount of structural information that are, in addition, difficult to interpret. Therefore, MD simulations are highly compatible with small-angle scattering experiments - simulations are used to interpret experimental data and in turn, experimental data are used to validate, and if necessary to guide simulations. Here, four different yet related questions are addressed. First, we quantify the influence of the ion cloud on interpreting SAXS data of charged proteins. Secondly, we study the size and the shape of detergent micelles, as this represents a starting point in improving the stability of protein-detergent complexes during the solubilization of membrane proteins. In the next step, we derive an ensemble of detergent micelles in agreement with experimental data, enabling us to study the in uence of various effects of SAXS curves and thereby making an important step towards the better understanding of the SAXS experimental data. Finally, we demonstrate how SAXS and SANS data can jointly be combined with MD simulations, allowing for fine structural characterization of proteindetergent complexes.Die akkurate Bestimmung makromolekularer Strukturen erfordert häufig experimentelle Daten mit rechnerischen Methoden zu kombinieren. In dieser Arbeit werden MD Simulationen zur Interpretation von experimentellen Daten, die mittels Kleinwinkel- Röntgenstreuung (SAXS) und Kleinwinkel-Neutronenstreuung (SANS) aufgenommen wurden, genutzt. SAXS- und SANS-Experimente werden unter nahezu natürlichen Bedingungen durchgeführt, liefern jedoch nur ein beschränktes Maß an struktureller Information, welche sich zudem auch nur schwer interpretieren lässt. MD Simulationen eignen sich besonders gut zur Kombination mit Kleinwinkelstreuexperimenten, da die Simulationen verwendet werden können, um experimentelle Daten zu interpretieren. Umgekehrt werden experimentelle Daten benutzt, um Simulationen zu validieren und sie nötigenfalls zu lenken. Im Folgenden werden vier verschiedene jedoch verbundene Fragestellungen behandelt. Im ersten Abschnitt wird der Ein uss einer lonenwolke auf die Interpretation von SAXS-Daten geladener Proteine untersucht. Danach wird die Form und Größe tensidbasierter Mizellen untersucht. Dies ist ein Ausgangspunkt, um die Stabilität von Protein-Detergenz Komplexen während des Lösens von Membranproteinen zu erhöhen. Im nächsten Schritt wird ein Ensemble tensidbasierter Mizellen bestimmt, welches mit expirmentellen Daten übereinstimmt und es ermöglicht, den Einfluss verschiedener Effekte auf die SAXS Kurve zu studieren. Dies stellt einen wichtigen Schritt dar, um experimentelle SAXS Daten besser zu verstehen. Als vierten Punkt demonstrieren wir, wie die Kombination von SAXS- und SANS-Daten zusammen mit MD-Simulation eine genaue Strukturbestimmung von Protein-Detergenz-Komplexen ermöglicht

Computational simulations on membranes and a transmembrane protein

To accurately model the transmembrane proteins, accurate descriptions of its natural environment, i.e., lipids, are critical. The all-atom CHARMM36 lipid force field (C36FF-AA) is tested with molecular dynamics (MD) simulations. Through comparison to experiments, we conclude that the C36FF-AA is accurate for use with bilayers of varying head and chain types over biologically relevant temperatures. The united-atom chain model of the C36FF (C36FF-UA) of common lipids is developed to improve simulation efficiency. It shows good agreement between the simulated bilayer properties obtained by C36FF-UA and experiments, and also between the simulated results from UA and AA lipid models. Besides the single-component membrane, multiple-components 18:2 linoleoyl-containing soybean membrane models have been developed. The structural properties of pure linoleoyl bilayers agree well with experiments, based on which the soybean membrane models also result in reasonable structural properties. Accurate lipid force field greatly facilitates the study of transmembrane proteins. Lactose permease of Escherichia coli (E. coli) belongs to major facilitator superfamily (MFS) which is the largest and most diverse family of transporters and serves as a model for secondary active transporters (SATs) in this dissertation. LacY structures of the cytoplasmic-open, occluded-like, and recently periplasmic-partially-open state have been determined, however, the crystal structure of LacY in the periplasmic-open state is still not available. The periplasmic-open LacY structure is important for understanding the complete proton/sugar transport process of LacY as well as other similar SAT proteins. MD simulations are performed to test the accuracy of the previously developed periplasmic-open LacYIM-EX model (JMB 404:506), and two other periplasmic-open LacY models, LacYSW and LacYFP models (JMB 407:698). The simulated results indicate that LacYIM-EX is the only structure that remains stable in the periplasmic-open state. The MD dummy spin label simulations (MDDS) have also been performed and the results show that the orientation of the spin labels significantly affect the distance measurement so that the proper interpretation of DEER requires the aid of MDDS simulations. Self-guided Langevin dynamics (SGLD) simulations are performed to search periplasmic-open LacY. The results show that no outward-facing is obtained with nanosecond-averaged results, but if we study individual structures, conformational sampling is obtained with certain SGLD parameters that enhance natural helical motions. This SGLD approach might hold promise for studying conformational changes of other SAT proteins

Computational Insights into the Antimicrobial Mechanism of Action of Class II Bacteriocins.

University of Minnesota Ph.D. dissertation May 2017. Major: Chemical Engineering. Advisor: Yiannis Kaznessis. 1 computer file (PDF); xi, 135 pages.Antibiotic resistance is a global problem and poses an alarming threat to public health. Microorganisms resistant to all commercially available antibiotics have emerged, undermining the ability to fight infectious diseases. The antibiotic resistance crisis has been attributed to the overuse of antibiotics, as well as a lack of new drug development. Coordinated efforts are needed to overcome this challenge, including discovery of alternative drugs. Bacteriocins are bacteria-produced, antimicrobial peptides that are potentially powerful antibiotic drug candidates. Despite considerable scientific interest around bacteriocins, and despite their promise as potent, latent antibiotics, their everyday medical value has been negligible. In order to more effectively utilize the full potential of bacteriocins as a platform to develop new antibacterial agents, a detailed understanding of their mechanism of action is required. This mechanistic insight will offer ways to control and optimize their activity and selectivity against specific pathogens, greatly enhancing their potential for medical applications. The goal of this work is to elucidate the mechanism of action of class II bacteriocins by employing a variety of computational methods that are built around atomistic molecular dynamics simulations. First, we studied Plantaricin EF, a two-peptide class IIb bacteriocin. This bacteriocin was simulated in different environments including water, micelles, and lipid bilayers. The interaction between the two peptides that promotes dimerization, and the interaction between the dimer and the membrane were elucidated. Guided by experimental studies, a transmembrane model of the dimer embedded in the bilayer was additionally designed. Results obtained from a 1 μs long atomistic molecular dynamics simulation, demonstrated for the first time that a bacteriocin, with a narrow antimicrobial activity range, can by itself form a water (and potentially ion) permeable, toroidal pore in a lipid bilayer. This pore was characterized in detail. It is not unlikely that the mechanism of action of bacteriocins can involve poration of the membrane as well as receptor-mediation. Therefore, the interaction of a bacteriocin with its putative receptor was also examined. Lacking the structure of a receptor, we employed structure-prediction techniques in combination with docking calculations, and molecular dynamics simulations. For the first time a class II bacteriocin-receptor complex was built, setting the ground for investigating the role that receptors play in the bactericidal activity of these antimicrobial peptides. We believe that our findings could be of importance to the designing of new antibiotic agents, as it would guide the search for better bacteriocins toward peptides with improved activity and specificity, that form stable pores, increase water or ion permeability, and interact more efficiently with a receptor