Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor.

No therapeutic drugs are currently available for nonalcoholic steatohepatitis (NASH) that progresses from nonalcoholic fatty liver via oxidative stress-involved pathways. Three cognate peroxisome proliferator-activated receptor (PPAR) subtypes (PPARα/δ/γ) are considered as attractive targets. Although lanifibranor (PPARα/δ/γ pan agonist) and saroglitazar (PPARα/γ dual agonist) are currently under investigation in clinical trials for NASH, the development of seladelpar (PPARδ-selective agonist), elafibranor (PPARα/δ dual agonist), and many other dual/pan agonists has been discontinued due to serious side effects or little/no efficacies. This study aimed to obtain functional and structural insights into the potency, efficacy, and selectivity against PPARα/δ/γ of three current and past anti-NASH investigational drugs: lanifibranor, seladelpar, and elafibranor. Ligand activities were evaluated by three assays to detect different facets of the PPAR activation: transactivation assay, coactivator recruitment assay, and thermal stability assay. Seven high-resolution cocrystal structures (namely, those of the PPARα/δ/γ-ligand-binding domain (LBD)-lanifibranor, PPARα/δ/γ-LBD-seladelpar, and PPARα-LBD-elafibranor) were obtained through X-ray diffraction analyses, six of which represent the first deposit in the Protein Data Bank. Lanifibranor and seladelpar were found to bind to different regions of the PPARα/δ/γ-ligand-binding pockets and activated all PPAR subtypes with different potencies and efficacies in the three assays. In contrast, elafibranor induced transactivation and coactivator recruitment (not thermal stability) of all PPAR subtypes, but the PPARδ/γ-LBD-elafibranor cocrystals were not obtained. These results illustrate the highly variable PPARα/δ/γ activation profiles and binding modes of these PPAR ligands that define their pharmacological actions.

[Toward High-throughput Crystal Structure Determination of PPAR Ligand-binding Domains in Complexes with Less Soluble Ligands].

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that are activated by endogenous fatty acids and synthetic compounds as ligands. We have been developing new phenylpropanoic acid derivatives based on structure-activity relationship studies that could reduce the side effects of existing clinical drugs. As a result, we have obtained many partial agonists that exhibit a moderate transcriptional activity while maintaining high specificity towards the receptors. However, because most of them are poorly soluble, protein-ligand interaction information has not yet been obtained by X-ray crystallography, which is essential for structure-activity relationship studies. In this paper, we report our ongoing crystallization experiments, which are aimed to develope a crystallization method for PPAR LBDs in solid-phase hydrogels that enables high-throughput protein-ligand complex crystal structure determination, using poorly soluble ligands.

Carboxyl Terminus of HOATZ is Intrinsically Disordered and Interacts with Heat Shock Protein A Families.

BACKGROUND: Hoatz is a vertebrate-specific gene, the defects of which result in hydrocephalus and oligo-astheno-teratozoospermia in mice. It encodes a 19-kDa protein lacking any domains of known function. METHODS: To understand the protein activity, we purified the carboxyl-terminal fragment that is conserved among different species, and analyzed its structure and potential binding proteins. A soluble 9.9-kDa HOATZ fragment, including a poly-histidine tag (designated HOATZ-C), was purified to homogeneity. RESULTS: The gel filtration profile and circular dichroism spectra collectively indicated that HOATZ-C was intrinsically disordered. When HOATZ-C was mixed with cleared lysate from Hoatz-null mouse testis, several proteins, including two of ~70 kDa size, were specifically co-purified with HOATZ-C on a nickel column. CONCLUSION: Based on the peptide mass fingerprinting of these bands, two members of the heat-shock protein family A were identified. These data may indicate the role of HOATZ in stress regulation in cells characterized by motile cilia and flagella.

Functional and Structural Insights into Human PPARα/δ/γ Subtype Selectivity of Bezafibrate, Fenofibric Acid, and Pemafibrate.

Among the agonists against three peroxisome proliferator-activated receptor (PPAR) subtypes, those against PPARα (fibrates) and PPARγ (glitazones) are currently used to treat dyslipidemia and type 2 diabetes, respectively, whereas PPARδ agonists are expected to be the next-generation metabolic disease drug. In addition, some dual/pan PPAR agonists are currently being investigated via clinical trials as one of the first curative drugs against nonalcoholic fatty liver disease (NAFLD). Because PPARα/δ/γ share considerable amino acid identity and three-dimensional structures, especially in ligand-binding domains (LBDs), clinically approved fibrates, such as bezafibrate, fenofibric acid, and pemafibrate, could also act on PPARδ/γ when used as anti-NAFLD drugs. Therefore, this study examined their PPARα/δ/γ selectivity using three independent assays-a dual luciferase-based GAL4 transactivation assay for COS-7 cells, time-resolved fluorescence resonance energy transfer-based coactivator recruitment assay, and circular dichroism spectroscopy-based thermostability assay. Although the efficacy and efficiency highly varied between agonists, assay types, and PPAR subtypes, the three fibrates, except fenofibric acid that did not affect PPARδ-mediated transactivation and coactivator recruitment, activated all PPAR subtypes in those assays. Furthermore, we aimed to obtain cocrystal structures of PPARδ/γ-LBD and the three fibrates via X-ray diffraction and versatile crystallization methods, which we recently used to obtain 34 structures of PPARα-LBD cocrystallized with 17 ligands, including the fibrates. We herein reveal five novel high-resolution structures of PPARδ/γ-bezafibrate, PPARγ-fenofibric acid, and PPARδ/γ-pemafibrate, thereby providing the molecular basis for their application beyond dyslipidemia treatment.

Pivotal role of a conserved histidine in Escherichia coli ribonuclease HI as proposed by X-ray crystallography.

The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme-Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme-Zn2+ complex a single metal ion was found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.

Crystal structures of the ligand-binding domain of human peroxisome proliferator-activated receptor δ in complexes with phenylpropanoic acid derivatives and a pyridine carboxylic acid derivative.

Peroxisome proliferator-activated receptor δ (PPARδ) is a member of the nuclear receptor family and regulates glucose and lipid homeostasis in a ligand-dependent manner. Numerous phenylpropanoic acid derivatives targeting three PPAR subtypes (PPARα, PPARγ and PPARδ) have been developed towards the treatment of serious diseases such as lipid-metabolism disorders. In spite of the increasing attraction of PPARδ as a pharmaceutical target, only a limited number of protein-ligand complex structures are available. Here, four crystal structures of the ligand-binding domain of PPARδ in complexes with phenylpropanoic acid derivatives and a pyridine carboxylic acid derivative are described, including an updated, higher resolution version of a previous studied structure and three novel structures. These structures showed that the ligands were bound in the ligand-binding pocket of the receptor in a similar manner but with minor variations. The results could provide variable structural information for the further design and development of ligands targeting PPARδ.

Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome.

Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN-MCM-GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD's DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N-Gins51C-GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

Crystal Structures of the Human Peroxisome Proliferator-Activated Receptor (PPAR)α Ligand-Binding Domain in Complexes with a Series of Phenylpropanoic Acid Derivatives Generated by a Ligand-Exchange Soaking Method.

Peroxisome proliferator-activated receptor (PPAR)α, a member of the nuclear receptor family, is a transcription factor that regulates the expression of genes related to lipid metabolism in a ligand-dependent manner, and has attracted attention as a target for hypolipidemic drugs. We have been developing phenylpropaonic acid derivatives as PPARα-targeted drug candidates for the treatment of metabolic diseases. Recently, we have developed the "ligand-exchange soaking method," which crystallizes the recombinant PPARα ligand-binding domain (LBD) as a complex with intrinsic fatty acids derived from an expression host Escherichia (E.) coli and thereafter replaces them with other higher-affinity ligands by soaking. Here we applied this method for preparation of cocrystals of PPARα LBD with its ligands that have not been obtained with the conventional cocrystallization method. We revealed the high-resolution structures of the cocrystals of PPARα LBD and the three synthetic phenylpropaonic acid derivatives: TIPP-703, APHM19, and YN4pai, the latter two of which are the first observations. The overall structures of cocrystals obtained from the two methods are identical and illustrate the close interaction between these ligands and the surrounding amino acid residues of PPARα LBD. This ligand-exchange soaking method could be applicable to high throughput preparations of co-crystals with another subtype PPARδ LBD for high resolution X-ray crystallography, because it also crystallizes in complex with intrinsic fatty acid(s) while not in the apo-form.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.

Preparation of co-crystals of human PPARα-LBD and ligand for high-resolution X-ray crystallography.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors with three subtypes (α, δ, and γ) that regulate cell differentiation and metabolism. Co-crystals of human PPARα-ligand-binding domain (LBD)-PPARα ligand for X-ray crystallography have been difficult to obtain. Recombinant human PPARα-LBD proteins contain intrinsic fatty acids (iFAs of Escherichia coli origin) and may be unstable without ligands during crystallization. To circumvent these limitations, we have successfully applied various crystallization techniques, including co-crystallization, cross-seeding, soaking, delipidation, and coactivator peptide supplementation. For complete details on the use and execution of this protocol, please refer to Kamata et al. (2020).

PPARα Ligand-Binding Domain Structures with Endogenous Fatty Acids and Fibrates.

Most triacylglycerol-lowering fibrates have been developed in the 1960s-1980s before their molecular target, peroxisome proliferator-activated receptor alpha (PPARα), was identified. Twenty-one ligand-bound PPARα structures have been deposited in the Protein Data Bank since 2001; however, binding modes of fibrates and physiological ligands remain unknown. Here we show thirty-four X-ray crystallographic structures of the PPARα ligand-binding domain, which are composed of a "Center" and four "Arm" regions, in complexes with five endogenous fatty acids, six fibrates in clinical use, and six synthetic PPARα agonists. High-resolution structural analyses, in combination with coactivator recruitment and thermostability assays, demonstrate that stearic and palmitic acids are presumably physiological ligands; coordination to Arm III is important for high PPARα potency/selectivity of pemafibrate and GW7647; and agonistic activities of four fibrates are enhanced by the partial agonist GW9662. These results renew our understanding of PPARα ligand recognition and contribute to the molecular design of next-generation PPAR-targeted drugs.

A crystallization and preliminary X-ray diffraction study of the Arabidopsis thaliana proliferating cell nuclear antigen (PCNA2) alone and in a complex with a PIP-box peptide from Flap endonuclease 1.

DNA replication is an important event for all living organisms and the mechanism is essentially conserved from archaea, bacteria to eukaryotes. Proliferating cell nuclear antigen (PCNA) acts as the universal platform for many DNA transacting proteins. Flap endonuclease 1 (FEN1) is one such enzyme whose activity is largely affected by the interaction with PCNA. To elucidate the key interactions between plant PCNA and FEN1 and possible structural change of PCNA caused by binding of FEN1 at the atomic level, crystallization and preliminary studies of X-ray diffraction of crystals of Arabidopsis thaliana PCNA2 (AtPCNA2) alone and in a complex with a peptide derived from AtFEN1, which contains a typical PCNA-interacting protein (PIP)-box motif, were performed. Both peptide-free and peptide-bound AtPCNA2s were crystallized using the same reservoir solution but in different crystal systems, indicating that the peptide affected the intermolecular interactions in the crystals. Crystals of AtPCNA2 belonged to the hexagonal space group P63, while those of the peptide-bound AtPCNA2 belonged to the rhombohedral space group H3, both of which could contain the functional homo-trimers.

Direct visualization of DNA baton pass between replication factors bound to PCNA.

In Eukarya and Archaea, the lagging strand synthesis is accomplished mainly by three key factors, DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig), in the DNA replication process. These three factors form important complexes with proliferating cell nuclear antigen (PCNA), thereby constructing a platform that enable each protein factor to act successively and smoothly on DNA. The structures of the Pol-PCNA-DNA and Lig-PCNA-DNA complexes alone have been visualized by single particle analysis. However, the FEN-PCNA-DNA complex structure remains unknown. In this report, we for the first time present this tertiary structure determined by single particle analysis. We also successfully visualized the structure of the FEN-Lig-PCNA-DNA complex, corresponding to a putative intermediate state between the removal of the DNA flap by FEN and the sealing of the nicked DNA by Lig. This structural study presents the direct visualization of the handing-over action, which proceeds between different replication factors on a single PCNA clamp bound to DNA. We detected a drastic conversion of the DNA from a bent form to a straight form, in addition to the dynamic motions of replication factors in the switching process.

Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication.

In eukaryotic DNA replication initiation, hexameric MCM (mini-chromosome maintenance) unwinds the template double-stranded DNA to form the replication fork. MCM is activated by two proteins, Cdc45 and GINS, which constitute the 'CMG' unwindosome complex together with the MCM core. The archaeal DNA replication system is quite similar to that of eukaryotes, but only limited knowledge about the DNA unwinding mechanism is available, from a structural point of view. Here, we describe the crystal structure of an archaeal GAN (GINS-associated nuclease) from Thermococcus kodakaraensis, the homolog of eukaryotic Cdc45, in both the free form and the complex with the C-terminal domain of the cognate Gins51 subunit (Gins51C). This first archaeal GAN structure exhibits a unique, 'hybrid' structure between the bacterial RecJ and the eukaryotic Cdc45. GAN possesses the conserved DHH and DHH1 domains responsible for the exonuclease activity, and an inserted CID (CMG interacting domain)-like domain structurally comparable to that in Cdc45, suggesting its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication. A structural comparison of the GAN-Gins51C complex with the GINS tetramer suggests that GINS uses the mobile Gins51C as a hook to bind GAN for CMG formation.

Integrated molecular mechanism directing nucleosome reorganization by human FACT.

Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular ß structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Peroxisome proliferator-activated receptor gamma (PPARγ) has multiple binding points that accommodate ligands in various conformations: Structurally similar PPARγ partial agonists bind to PPARγ LBD in different conformations.

In the course of studies directed toward the creation of human peroxisome proliferator-activated receptor gamma (hPPARγ) partial agonists, we designed and synthesized benzylsulfonylaminocarbonyl derivative (3) by structural modification of our reported hPPARγ partial agonist 2. Co-crystallization of 3 with the hPPARγ ligand-binding domain (LBD) afforded a homodimeric complex in which one of the LBDs adopts a fully active structure without bound 3, while the other LBD exhibits a non-fully active structure containing one molecule of bound 3. Interestingly, 2 and 3 are structurally similar, but bind to hPPARγ LBD in distinct conformations, that is, the sulfonylaminocarbonyl moiety of bound 3 is directed at 180° away from that of bound 2. These results support our previous proposal that the hPPARγ LBD has multiple binding points that can be utilized to accommodate structurally flexible hPPAR ligands.

Different structures of the two peroxisome proliferator-activated receptor gamma (PPARγ) ligand-binding domains in homodimeric complex with partial agonist, but not full agonist.

We designed and synthesized acylsulfonamide derivative (3) as a human peroxisome proliferator-activated receptor gamma (hPPARγ) partial agonist by structural modification of hPPARγ full agonist 1. Co-crystallization of 3 with hPPARγ LBD afforded a homodimeric complex, and X-ray crystallographic analysis at 2.1Šresolution showed that one of the LBDs adopts a fully active structure identical with that in the complex of rosiglitazone, a full agonist; however, the other LBD in the complex of 3 exhibits a different (non-fully active) structure. Interestingly, the apo-homodimer contained similar LBD structures. Intrigued by these results, we surveyed reported X-ray crystal structures of partial agonists complexed with hPPARγ LBD homodimer, and identified several types of LBD structures distinct from the fully active structure. In contrast, both LBDs in the rosiglitazone complex have the fully active structure. These results suggest hPPARγ partial agonists lack the ability to induce fully active LBD. The presence of at least one non-fully active LBD in the agonist complex may be a useful criterion to distinguish hPPARγ partial agonists from full agonists.

Structural design and synthesis of arylalkynyl amide-type peroxisome proliferator-activated receptor γ (PPARγ)-selective antagonists based on the helix12-folding inhibition hypothesis.

Peroxisome proliferator-activated receptor γ (PPARγ) antagonists are candidates for treatment of type 2 diabetes, obesity and osteoporosis. However, few rational design strategies are currently available. Here, we utilized the helix12 (H12)-folding inhibition hypothesis, in combination with our previously determined X-ray crystal structure of PPARγ agonist MEKT-21 (6) complexed with the PPARγ ligand-binding domain, to design and develop a potent phenylalkynyl amide-type PPARγ antagonist 9i, focusing initially on pinpoint structural modification of the propanoic acid moiety of 6. Since 9i retained very weak, but distinct, PPARγ agonist activity, we next modified the distal benzene ring of 9i, aiming to delete the residual PPARγ agonist activity while retaining the antagonist activity. Introduction of a chlorine atom at the 2-position of the distal benzene ring afforded 9p, which exhibited potent, PPARγ-selective full antagonist activity without detectable agonist activity. We found that 9p stabilized the corepressor-PPARγ complex and suppressed basal PPARγ activity. This compound showed anti-adipogenesis activity at the cellular level. This agonist-antagonist switching concept based on the H12-folding inhibition hypothesis should also be applicable for designing other classes of PPARγ full antagonists.

Disordered interdomain region of Gins is important for functional tetramer formation to stimulate MCM helicase in Thermoplasma acidophilum.

The eukaryotic MCM is activated by forming the CMG complex with Cdc45 and GINS to work as a replicative helicase. The eukaryotic GINS consists of four different proteins to form tetrameric complex. In contrast, the TaGins51 protein from the thermophilic archaeon, Thermoplasma acidophilum forms a homotetramer (TaGINS), and interacts with the cognate MCM (TaMCM) to stimulate the DNA-binding, ATPase, and helicase activities of TaMCM. All Gins proteins from Archaea and Eukarya contain α-helical A- and ß-stranded B-domains. Here, we found that TaGins51 forms the tetramer without the B-domain. However, the A-domain without the linker region between the A- and B-domains could not form a stable tetramer, and furthermore, the A-domain by itself could not stimulate the TaMCM activity. These results suggest that the formation of the Gins51 tetramer is necessary for MCM activation, and the disordered linker region between the two domains is critical for the functional complex formation.

Structure of the entire stalk region of the Dynein motor domain.

Dyneins are large microtubule-based motor complexes that power a range of cellular processes including the transport of organelles, as well as the beating of cilia and flagella. The motor domain is located within the dynein heavy chain and comprises an N-terminal mechanical linker element, a central ring of six AAA+ modules of which four bind or hydrolyze ATP, and a long stalk extending from the AAA+ring with a microtubule-binding domain (MTBD) at its tip. A crucial mechanism underlying the motile activity of cytoskeletal motor proteins is precise coupling between the ATPase and track-binding activities. In dynein, a stalk region consisting of a long (~15nm) antiparallel coiled coil separates these two activities, which must facilitate communication between them. This communication is mediated by a small degree of helix sliding in the coiled coil. However, no high-resolution structure is available of the entire stalk region including the MTBD. Here, we have reported the structure of the entire stalk region of mouse cytoplasmic dynein in a weak microtubule-binding state, which was determined using X-ray crystallography, and have compared it with the dynein motor domain from Dictyostelium discoideum in a strong microtubule-binding state and with a mouse MTBD with its distal portion of the coiled coil fused to seryl-tRNA synthetase from Thermus thermophilus. Our results strongly support the helix-sliding model based on the complete structure of the dynein stalk with a different form of coiled-coil packing. We also propose a plausible mechanism of helix sliding together with further analysis using molecular dynamics simulations. Our results present the importance of conserved proline residues for an elastic motion of stalk coiled coil and imply the manner of change between high-affinity state and low-affinity state of MTBD.