A Hybrid Nanotube Stamp System in Intracellular Protein Delivery for Cancer Treatment and NMR Analytical Techniques.

In contrast to intracellular gene transfer, the direct delivery of expressed proteins is a significantly challenging yet essential technique for elucidating cellular functions, including protein complex structure, liquid-liquid phase separation, therapeutic applications, and reprogramming. In this study, we developed a hybrid nanotube (HyNT) stamp system that physically inserts the HyNTs into adhesive cells, enabling the injection of target molecules through HyNT ducts. This system demonstrates the capability to deliver multiple proteins, such as lactate oxidase (LOx) and ubiquitin (UQ), to approximately 1.8 × 107 adhesive cells with a delivery efficiency of 89.9% and a viability of 97.1%. The delivery of LOx enzyme into HeLa cancer cells induced cell death, while enzyme-delivered healthy cells remained viable. Furthermore, our stamp system can deliver an isotope-labeled UQ into adhesive cells for detection by nuclear magnetic resonance (NMR).

Activation of L-lactate oxidase by the formation of enzyme assemblies through liquid-liquid phase separation.

The assembly state of enzymes is gaining interest as a mechanism for regulating the function of enzymes in living cells. One of the current topics in enzymology is the relationship between enzyme activity and the assembly state due to liquid-liquid phase separation. In this study, we demonstrated enzyme activation via the formation of enzyme assemblies using L-lactate oxidase (LOX). LOX formed hundreds of nanometer-scale assemblies with poly-L-lysine (PLL). In the presence of ammonium sulfate, the LOX-PLL clusters formed micrometer-scale liquid droplets. The enzyme activities of LOX in clusters and droplets were one order of magnitude higher than those in the dispersed state, owing to a decrease in KM and an increase in kcat. Moreover, the clusters exhibited a higher activation effect than the droplets. In addition, the conformation of LOX changed in the clusters, resulting in increased enzyme activation. Understanding enzyme activation and assembly states provides important information regarding enzyme function in living cells, in addition to biotechnology applications.

Identification of a Proline-Kinked Amphipathic α-Helix Downstream from the Methyltransferase Domain of a Potexvirus Replicase and Its Role in Virus Replication and Perinuclear Complex Formation.

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus, the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of Plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain, and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. Nuclear magnetic resonance (NMR) analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a green fluorescent protein (GFP) fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA colocated during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions, and in vitro high-molecular-weight complexes were formed by both replication-competent and -incompetent viral replicons and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales. Substitution of the proline residue, but not the other residues necessary for replication, allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.

Physicochemical characterization of the G51D mutation of α-synuclein that is responsible for its severe cytotoxicity.

Fibril formation and aggregation of α-synuclein are important for the pathogenesis of neurodegenerative disorders including Parkinson's disease. In familial Parkinson's disease, the G51D mutation of α-synuclein causes severe symptoms and rapid progression. α-Synuclein, an intrinsically disordered protein, was shown to adopt an α-helical tetrameric state that resists fibrillation and aggregation. Here, we isolated the stable dimeric state of recombinant wild-type (WT) α-synuclein and G51D α-synuclein protein. Using circular dichroism spectroscopy, we determined that the α-synuclein dimer and monomer structures were unfolded. The WT α-synuclein dimer was more resistant to fibril formation than the monomer. However, the fibril formation rate of the G51D α-synuclein dimer was similar to that of the G51D α-synuclein monomer. The fibril morphology and properties of the G51D α-synuclein monomer were different from those of the WT α-synuclein monomer and dimer and G51D α-synuclein dimer. Additionally, G51D α-synuclein monomer fibrils were more cytotoxic than other fibrils. Our findings indicate that the structural differences between G51D α-synuclein monomer fibrils and other fibrils are critically responsible for its severe neurotoxicity in familial Parkinson's disease.

Ultrasensitive Change in Nucleosome Binding by Multiple Phosphorylations to the Intrinsically Disordered Region of the Histone Chaperone FACT.

Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

Effect of site-specific amino acid D-isomerization on ß-sheet transition and fibril formation profiles of Tau microtubule-binding repeat peptides.

d-amino acid-containing proteins have been found in several human tissues, and the spontaneous accumulation of d-amino acids in proteins is thought to be involved in age-dependent diseases including dementia. Tau, a microtubule-associated protein, is a major component of neurofibrillary tangles in Alzheimer's disease. Site-specific amino acid D-isomerization in Tau has been observed in the brains of patients with Alzheimer's disease. Here, we conducted amino acid D-isomerization at specific sites in microtubule-binding repeat peptides of Tau (Tau R2 and R3) and examined the effects on Tau structure and fibril formation. Our results demonstrate that amino acid D-isomerization in Tau R2 peptides decreased the rates of ß-sheet transition and fibril formation compared with those of the wild-type peptide composed of all l-amino acids. In contrast, Tau R3 peptides that had undergone amino acid D-isomerization at either Asp314, Ser316, or Ser324 showed increased rates of ß-sheet transition and fibril formation compared with those of the wild-type Tau R3 peptide.

The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation.

In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.

Substrate Binding Switches the Conformation at the Lynchpin Site in the Substrate-Binding Domain of Human Hsp70 to Enable Allosteric Interdomain Communication.

The stress-induced 70 kDa heat shock protein (Hsp70) functions as a molecular chaperone to maintain protein homeostasis. Hsp70 contains an N-terminal ATPase domain (NBD) and a C-terminal substrate-binding domain (SBD). The SBD is divided into the ß subdomain containing the substrate-binding site (ßSBD) and the α-helical subdomain (αLid) that covers the ßSBD. In this report, the solution structures of two different forms of the SBD from human Hsp70 were solved. One structure shows the αLid bound to the substrate-binding site intramolecularly, whereas this intramolecular binding mode is absent in the other structure solved. Structural comparison of the two SBDs from Hsp70 revealed that client-peptide binding rearranges residues at the interdomain contact site, which impairs interdomain contact between the SBD and the NBD. Peptide binding also disrupted the inter-subdomain interaction connecting the αLid to the ßSBD, which allows the binding of the αLid to the NBD. The results provide a mechanism for interdomain communication upon substrate binding from the SBD to the NBD via the lynchpin site in the ßSBD of human Hsp70. In comparison to the bacterial ortholog, DnaK, some remarkable differences in the allosteric signal propagation among residues within the Hsp70 SBD exist.

Dynamic Allostery Modulates Catalytic Activity by Modifying the Hydrogen Bonding Network in the Catalytic Site of Human Pin1.

Allosteric communication among domains in modular proteins consisting of flexibly linked domains with complimentary roles remains poorly understood. To understand how complementary domains communicate, we have studied human Pin1, a representative modular protein with two domains mutually tethered by a flexible linker: a WW domain for substrate recognition and a peptidyl-prolyl isomerase (PPIase) domain. Previous studies of Pin1 showed that physical contact between the domains causes dynamic allostery by reducing conformation dynamics in the catalytic domain, which compensates for the entropy costs of substrate binding to the catalytic site and thus increases catalytic activity. In this study, the S138A mutant PPIase domain, a mutation that mimics the structural impact of the interdomain contact, was demonstrated to display dynamic allostery by rigidification of the α2-α3 loop that harbors the key catalytic residue C113. The reduced dynamics of the α2-α3 loop stabilizes the C113-H59 hydrogen bond in the hydrogen-bonding network of the catalytic site. The stabilized hydrogen bond between C113 and H59 retards initiation of isomerization, which explains the reduced isomerization rate by ~20% caused by the S138A mutation. These results provide new insight into the interdomain allosteric communication of Pin1.

Molecular actions of two synthetic brassinosteroids, iso-carbaBL and 6-deoxoBL, which cause altered physiological activities between Arabidopsis and rice.

Brassinosteroid (BR) is an important plant hormone that is perceived by the BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor. BRI1 is conserved among dicot and monocot species; however, the molecular mechanism underlying BR perception in monocots is not fully understood. We synthesised two BRs, iso-carbabrassinolide (iso-carbaBL) and 6-deoxoBL, which have different BR activities in Arabidopsis thaliana (Arabidopsis) and rice. Our bioassay indicated that iso-carbaBL has relatively strong BR activity in Arabidopsis, but is inactive in rice and competitively inhibits BR activity. The bioactivity of 6-deoxoBL was similar to that of BL in Arabidopsis, but was much lower in rice. Binding experiments using recombinant Arabidopsis and rice BRI1 protein fragments suggested that iso-carbaBL and 6-deoxoBL bind to both receptors. These results showed that iso-carbaBL and 6-deoxoBL act as an antagonist and agonist, respectively, of BRs in rice. A docking simulation analysis suggested that iso-carbaBL fits deeper in the binding pocket to block the binding of active BR to rice BRI1. The simulated binding energy of 6-deoxoBL with rice BRI1 is much lower than that with Arabidopsis BRI1. The possible structural characteristics of rice BRI1 were determined based on the difference in the BR activities of iso-carbaBL and 6-deoxoBL in Arabidopsis and rice.

Solution structure of the first RNA recognition motif domain of human spliceosomal protein SF3b49 and its mode of interaction with a SF3b145 fragment.

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598-631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of ß1-α1-ß2-ß3-α2-ß4. Furthermore, a docking model based on NOESY measurements suggests that residues 607-616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix-helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull-down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix-helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145.

Hesperidin interaction to collagen detected by physico-chemical techniques.

OBJECTIVE: Dentin collagen can be modified by some plant-derived flavonoids to improve properties of dentin organic matrix. Hesperidin (HPN), a hesperetin-7-O-rutinoside flavonoid, has a potential of dentin modification for being based on evidence that a treatment with HPN may resist collagenase degradation and arrest demineralization of human dentin. In this study, biophysical and molecular-level information on the interaction of HPN and collagen was investigated. METHODS: HPN is extracted from citrus fruits. Sample collagenous solution was prepared using atelocollagen (ATCL) as a triple-helical peptide model. We have performed circular dichroism spectroscopic analysis, sedimentation velocity measurement by ultracentrifuge and saturation transfer difference measurement (STD) by NMR on HPN-collagen in solution state. RESULTS: The circular dichroism and sedimentation velocity measurement showed the evidence for the molecular interaction between ATCL and HPN, while HPN did not induce any conformational change of ATCL. The STD-NMR study further confirmed this interaction and suggested that HPN interacted with ATCL through its aromatic part, not through its disaccharide moiety. SIGNIFICANCE: These findings indicated that HPN is weakly bound to ATCL not causing structural modification of collagen. This interaction may contribute to the preservation of collagen by protecting from collagenase degradation.

A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

Angucycline antibiotic waldiomycin recognizes common structural motif conserved in bacterial histidine kinases.

Two-component signal transduction systems (TCSs), composed of a histidine kinase sensor (HK) and its cognate response regulator, sense and respond to environmental changes and are related to the virulence of pathogens. TCSs are potential targets for alternative antibiotics and anti-virulence agents. Here we found that waldiomycin, an angucycline antibiotic that inhibits a growth essential HK, WalK, in Gram-positive bacteria, also inhibits several class I HKs from the Gram-negative Escherichia coli. NMR analyses and site-directed mutagenesis studies using the osmo-sensing EnvZ, a prototypical HK of E. coli, showed that waldiomycin directly binds to both H-box and X-region, which are the two conserved regions in the dimerization-inducing and histidine-containing phosphotransfer (DHp) domain of HKs. Waldiomycin inhibits phosphorylation of the conserved histidine in the H-box. Analysis of waldiomycin derivatives suggests that the angucyclic ring, situated near the H-box in the waldiomycin-EnvZ DHp domain complex model, is responsible for the inhibitory activity. We demonstrate that waldiomycin is an HK inhibitor binding to the H-box region and has the potential of inhibiting a broad spectrum of HKs.

Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy.

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.

Allosteric Breakage of the Hydrogen Bond within the Dual-Histidine Motif in the Active Site of Human Pin1 PPIase.

Intimate cooperativity among active site residues in enzymes is a key factor for regulating elaborate reactions that would otherwise not occur readily. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is the phosphorylation-dependent cis-trans peptidyl-prolyl isomerase (PPIase) that specifically targets phosphorylated Ser/Thr-Pro motifs. Residues C113, H59, H157, and T152 form a hydrogen bond network in the active site, as in the noted connection. Theoretical studies have shown that protonation to thiolate C113 leads to rearrangement of this hydrogen bond network, with switching of the tautomeric states of adjacent histidines (H59 and H157) [Barman, A., and Hamelberg, D. (2014) Biochemistry 53, 3839-3850]. This is called the "dual-histidine motif". Here, C113A and C113S Pin1 mutants were found to alter the protonation states of H59 according to the respective residue type replaced at C113, and the mutations resulted in disruption of the hydrogen bond within the dual-histidine motif. In the C113A mutant, H59 was observed to be in exchange between ε- and δ-tautomers, which widened the entrance of the active site cavity, as seen by an increase in the distance between residues A113 and S154. The C113S mutant caused H59 to exchange between the ε-tautomer and imidazolium while not changing the active site structure. Moreover, the imidazole ring orientations of H59 and H157 were changed in the C113S mutant. These results demonstrated that a mutation at C113 modulates the hydrogen bond network dynamics. Thus, C113 acts as a pivot to drive the concerted function among the residues in the hydrogen bond network, as theoretically predicted.

Solution structures of the DNA-binding domains of immune-related zinc-finger protein ZFAT.

ZFAT is a transcriptional regulator, containing eighteen C2H2-type zinc-fingers and one AT-hook, involved in autoimmune thyroid disease, apoptosis, and immune-related cell survival. We determined the solution structures of the thirteen individual ZFAT zinc-fingers (ZF) and the tandemly arrayed zinc-fingers in the regions from ZF2 to ZF5, by NMR spectroscopy. ZFAT has eight uncommon bulged-out helix-containing zinc-fingers, and six of their structures (ZF4, ZF5, ZF6, ZF10, ZF11, and ZF13) were determined. The distribution patterns of the putative DNA-binding surface residues are different among the ZFAT zinc-fingers, suggesting the distinct DNA sequence preferences of the N-terminal and C-terminal zinc-fingers. Since ZFAT has three to five consecutive tandem zinc-fingers, which may cooperatively function as a unit, we also determined two tandemly arrayed zinc-finger structures, between ZF2 to ZF4 and ZF3 to ZF5. Our NMR spectroscopic analysis detected the interaction between ZF4 and ZF5, which are connected by an uncommon linker sequence, KKIK. The ZF4-ZF5 linker restrained the relative structural space between the two zinc-fingers in solution, unlike the other linker regions with determined structures, suggesting the involvement of the ZF4-ZF5 interfinger linker in the regulation of ZFAT function.

Molecular level evaluation on HEMA interaction with a collagen model.

OBJECTIVE: 2-Hydroxyethylmethacrylate (HEMA) diffuses in wet dentin and promotes adhesion during dentin priming and bonding. We have investigated the molecular level interaction between HEMA and a collagen model by using saturation transfer difference (STD) NMR. METHODS: The binding of HEMA to collagen was preliminarily investigated by suspending demineralized human dentin powders in a 4mM HEMA solution for 1h and measuring the decrease in the HEMA concentration on a spectrophotometer. The molecular level interaction of HEMA with atelocollagen, which was used as a collagen model, was investigated by STD-NMR spectroscopy. RESULTS: The HEMA concentration in the suspension did not change, indicating that HEMA did not bind to dentin collagen. This was confirmed by STD-NMR; when the atelocollagen resonance was saturated, no saturation was propagated to HEMA and no STD signals were detected. SIGNIFICANCE: The HEMA protons were not near the atelocollagen surface, indicating HEMA did not interact with atelocollagen. The collagen fibrils may be surrounded by water molecules in dentin/bond interfaces, which prevent the direct HEMA binding interaction.

Comparison of residual alpha- and beta-structures between two intrinsically disordered proteins by using NMR.

Intrinsically disordered proteins contain some residual structures, which may fold further upon binding to the partner protein for function. The residual structures observed in two intrinsically disordered proteins, including the C-terminal segment of peripherin-2 (63 residues) and measles virus nucleocapsid protein Ntail (125 residues), were compared using NMR. Differences in the chemical shifts of alpha-, beta- and carbonyl carbons between the observed structure and calculated random coil revealed the existence of a helix and some possible beta-structures in both proteins. The intensity of signals in the C-terminal segment of peripherin-2 in NMR spectra was informative and locally low, particularly in the middle and N-terminal parts: this suggested the broadening of the signals caused by the formation of residual structures in those areas. Furthermore, the protection of exchange of amide protons was significantly observed at the N-terminus. Conversely, the intensities of signals for Ntail were random beyond the overall areas of protein, and indicated no characteristic pattern. Only a faint protection of amide-proton exchange in Ntail was observed in the C-terminus. It was concluded that Ntail was more intrinsically disordered than the C-terminal segment of peripherin-2. The combination of chemical shifts with the amide-proton exchanges and signal intensities was useful for the analyses of the remaining secondary structures. The beta-structure might be more detectable by the protection of amide-proton exchange than the helical structure, although the changes in chemical shifts were sensitive for the detection of elements of both secondary structures.

The C113D mutation in human Pin1 causes allosteric structural changes in the phosphate binding pocket of the PPIase domain through the tug of war in the dual-histidine motif.

Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes specifically the pSer/pThr-Pro motif. The cis-trans isomerization mechanism has been studied by various approaches, including X-ray crystallography, site-directed mutagenesis, and the kinetic isotope effect on isomerization. However, a complete picture of the reaction mechanism remains elusive. On the basis of the X-ray structure of Pin1, residue C113 was proposed to play a nucleophile attacker to catalyze the isomerization. The controversial result that the C113D Pin1 mutant retains the activity, albeit at a reduced level, challenges the importance of C113 as a catalyst. To facilitate our understanding of the Pin1 isomerization process, we compared the structures and dynamics of the wild type with those of the C113D mutant Pin1 PPIase domains (residues 51-163). We found the C113D mutation disturbed the hydrogen bonds between the conserved histidine residues, H59 and H157 ("dual-histidine motif"); H59 imidazole forms a stable hydrogen bond to H157 in the wild type, whereas it has a strong hydrogen bond to D113 with weakened bonding to H157 in the C113D mutant. The C113D mutation unbalanced the hydrogen bonding tug of war for H59 between C113/D113 and H157 and destabilized the catalytic site structure, which eventually resulted in an altered conformation of the basic triad (K63, R68, and R69) that binds to the phosphate group in a substrate. The change in the basic triad structure could explain the severely weakened substrate binding ability of the C113D mutant. Overall, this work demonstrated that C113 plays a role in keeping the catalytic site in an active fold, which has never before been described.