Intermolecular Interface Research Articles

Ultrasound treatment improved gelling and emulsifying properties of myofibrillar proteins from Antarctic krill (Euphausia superba)

Antarctic krill is a promising source of marine proteins with abundant biomass and excellent nutritional profile, but has poor technological properties. Ultrasonic treatment at power levels of 0, 100, 200, 300, 400 and 500 W was applied to improve the technological properties of Antarctic krill meat, and the changes in physicochemical properties of myofibrillar proteins (MPs) were investigated. The results indicated that proper ultrasonic treatment significantly improved the gelling properties of Antarctic krill meat, in terms of a more uniform and stable gel texture and better water holding capacity, which were related to better cross-linking of MPs. Ultrasonic treatment promoted the conversion of MPs’ secondary structures from α-helix and random coil to β-sheet and β-turn, thereby making the molecular structure soft and loose. In addition, at tertiary structure level, ultrasonic treatment exposed the hydrophobic groups and sulfhydryl groups within MPs, thereby improving the emulsifying properties by changing the intermolecular interactions and interface properties. Furthermore, the particle size of MPs decreased and exhibited a more uniform distribution, aligning with the enhanced interactions observed between MPs and oil. These results provide an insight into the efficient development of Antarctic krill by elucidating how the ultrasonic treatment improves the gelling and emulsifying properties based on structure modulation of myofibrillar proteins.

Yop1 stability and membrane curvature generation propensity are controlled by its oligomerisation interface.

The DP1 family of integral membrane proteins stabilize high membrane curvature in the endoplasmic reticulum and phagophores. Mutations in the human DP1 gene REEP1 are associated with Hereditary Spastic Paraplegia type 31 and distal hereditary motor neuropathy. Four missense mutations map to a putative dimerization interface but the impact of these mutations on DP1 structure and tubule formation are unknown. Combining biophysical measurements, functional assays, and computational modeling in the context of the model protein Yop1, we found that missense mutations have variable effects on DP1 dimer structure and in vitro tubulation activity, and provide mechanistic insights into the role of DP1 oligomerisation on membrane curvature stabilization. Whereas the mutations P71L and S75F decreased dimer homogeneity and led to polydisperse oligomerization and impaired membrane curving activity, A72E introduced new polar interactions between subunits that stabilized the Yop1 dimer and allowed robust tubule formation but prevented formation of more highly-curved lipoprotein particles (LPP). The introduction of a BRIL domain to the cytoplasmic loop of A72E rescued LPP formation, consistent with a requirement for dimer splaying in highly curved membranes. These results suggest that the membrane curving activity of DP1 proteins requires both dimer stability and conformational plasticity at the intermolecular interface.

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

The role of disulfide bonds in the formation of the spatial structure of the human epidermal growth factor

The epidermal growth factor (EGF) is a peptide of the EGF-like growth factor family with a common conserved EGF-like domain formed by three intramolecular disulfide bonds. This article describes changes in the spatial structure of EGF and its mutant form with the D46G substitution in its C-terminal fragment observed upon disulfide bonds reduction in the corresponding synthetic peptides in a 0.01 M phosphate buffer (pH = 7.4). The structure was analyzed using circular dichroism spectroscopy, spectrofluorimetry, native polyacrylamide gel electrophoresis, and centrifugal ultrafiltration. It was shown that disulfide bonds reduction changes the geometry of the EGF-like domain towards an increase in the content of the beta-structure, while these peptides remain in dimeric form. According to the molecular modeling results, this can lead to the elongation of the main beta-hairpin of the EGF-like domain, to the elongation of the intermolecular beta-structure, or to the formation of a new beta-structure between the N- and C-terminal fragments of each molecule, which will change the intermolecular interface in dimeric form. Disulfide bonds reduction prevents EGF dimer dissociation to monomers. Under physiological conditions, this can lead to the inability of EGF to form binding sites for EGFR (epidermal growth factor receptor) and to cause its activation.

HINT, a code for understanding the interaction between biomolecules: a tribute to Donald J. Abraham.

A long-lasting goal of computational biochemists, medicinal chemists, and structural biologists has been the development of tools capable of deciphering the molecule-molecule interaction code that produces a rich variety of complex biomolecular assemblies comprised of the many different simple and biological molecules of life: water, small metabolites, cofactors, substrates, proteins, DNAs, and RNAs. Software applications that can mimic the interactions amongst all of these species, taking account of the laws of thermodynamics, would help gain information for understanding qualitatively and quantitatively key determinants contributing to the energetics of the bimolecular recognition process. This, in turn, would allow the design of novel compounds that might bind at the intermolecular interface by either preventing or reinforcing the recognition. HINT, hydropathic interaction, was a model and software code developed from a deceptively simple idea of Donald Abraham with the close collaboration with Glen Kellogg at Virginia Commonwealth University. HINT is based on a function that scores atom-atom interaction using LogP, the partition coefficient of any molecule between two phases; here, the solvents are water that mimics the cytoplasm milieu and octanol that mimics the protein internal hydropathic environment. This review summarizes the results of the extensive and successful collaboration between Abraham and Kellogg at VCU and the group at the University of Parma for testing HINT in a variety of different biomolecular interactions, from proteins with ligands to proteins with DNA.

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a β-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

SynDLP is a dynamin-like protein of Synechocystis sp. PCC 6803 with eukaryotic features

Dynamin-like proteins are membrane remodeling GTPases with well-understood functions in eukaryotic cells. However, bacterial dynamin-like proteins are still poorly investigated. SynDLP, the dynamin-like protein of the cyanobacterium Synechocystis sp. PCC 6803, forms ordered oligomers in solution. The 3.7 Å resolution cryo-EM structure of SynDLP oligomers reveals the presence of oligomeric stalk interfaces typical for eukaryotic dynamin-like proteins. The bundle signaling element domain shows distinct features, such as an intramolecular disulfide bridge that affects the GTPase activity, or an expanded intermolecular interface with the GTPase domain. In addition to typical GD-GD contacts, such atypical GTPase domain interfaces might be a GTPase activity regulating tool in oligomerized SynDLP. Furthermore, we show that SynDLP interacts with and intercalates into membranes containing negatively charged thylakoid membrane lipids independent of nucleotides. The structural characteristics of SynDLP oligomers suggest it to be the closest known bacterial ancestor of eukaryotic dynamin.

Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14

Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn2+-containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging. Here, in an effort to improve MMP specificity, we incorporated the metal-binding non-canonical amino acids (NCAAs), 3,4-dihydroxyphenylalanine (L-DOPA) and (8-hydroxyquinolin-3-yl)alanine (HqAla), into the MMP-inhibitory N-terminal domain of TIMP2 (N-TIMP2) at selected positions that interact with the catalytic Zn2+ ion (S2, S69, A70, L100) or with a structural Ca2+ ion (Y36). Evaluation of the inhibitory potency of the NCAA-containing variants towards MMP-2, MMP-9 and MMP-14 in vitro revealed that most showed a significant loss of inhibitory activity towards MMP-14, but not towards MMP-2 and MMP-9, resulting in increased specificity towards the latter proteases. Substitutions at S69 conferred the best improvement in selectivity for both L-DOPA and HqAla variants. Molecular modeling provided an indication of how MMP-2 and MMP-9 are better able to accommodate the bulky NCAA substituents at the intermolecular interface with N-TIMP2. The models also showed that, rather than coordinating to Zn2+, the NCAA side chains formed stabilizing polar interactions at the intermolecular interface with MMP-2 and MMP-9. Our findings illustrate how incorporation of NCAAs can be used to probe—and possibly exploit—differential tolerance for substitution within closely related protein–protein complexes as a means to improve specificity.

Structure of human placental steroid sulfatase at 2.0 angstrom resolution: Catalysis, quaternary association, and a secondary ligand site

Human placental estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (human placental steroid sulfatase; hSTS) is an integral membrane protein of the endoplasmic reticulum. This Ca2+-dependent enzyme catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, which then act as precursors for the biosynthesis of 17β-estradiol (E2) and dihydrotestosterone (DHT), respectively, the most potent forms of estrogens and androgens. hSTS is a key enzyme for the local production of E2 and DHT in the breast and the prostate. The enzyme is known to be responsible for maintaining high levels of estrogens in the breast tumor cells. The crystal structure of hSTS purified from human placenta has previously been reported at 2.6 Å resolution. Here we present the structure of hSTS determined at the superior 2.0 Å resolution bringing new clarity to the atomic architecture of the active site. The molecular basis of catalysis and steroid-protein interaction are revisited in light of the new data. We also reexamine the enzyme’s quaternary association and its implication on the membrane integration. A secondary ligand binding pocket at the intermolecular interface and adjacent to the active site access channel, buried into the gill of the mushroom-shaped molecule, has been identified. Its role as well as that of a phosphate ion bound to an exposed histidine side chain are examined from the structure-function perspective. Higher resolution data also aids in the tracing of an important loop missing in the previous structure.

Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution

An interplay between monomeric and dimeric forms of human epidermal growth factor (EGF) affecting its interaction with EGF receptor (EGFR) is poorly understood. While EGF dimeric structure was resolved at pH 8.1, the possibility of EGF dimerization under physiological conditions is still unclear. This study aimed to describe the oligomeric state of EGF in a solution at physiological pH value. With centrifugal ultrafiltration followed by blue native gel electrophoresis, we showed that synthetic human EGF in a solution at a concentration of 0.1mg/ml exists mainly in the dimeric form at pH 7.4 and temperature of 37°C, although a small fraction of its monomers was also observed. Based on bioinformatics predictions, we introduced the D46G substitution to examine if EGF C-terminal part is directly involved in the intermolecular interface formation of the observed dimers. We found a reduced ability of the resulting EGF D46G dimers to dissociate at temperatures up to 50°C. The D46G substitution also increased the intermolecular antiparallel β-structure content within the EGF peptide in a solution according to the CD spectra analysis that was confirmed by HATR-FTIR results. Additionally, the energy transfer between Tyr and Trp residues was detected by fluorescence spectroscopy for the EGF D46G mutant, but not for the native EGF. This allowed us to suggest the elongation and rearrangement of the intermolecular β-structure that leads to the observed stabilization of EGF D46G dimers. The results imply EGF dimerization under physiological pH value and temperature and the involvement of EGF C-terminal part in this process.

Surface Engineering of Top7 to Facilitate Structure Determination.

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

Domain Organization of the UBX Domain Containing Protein 9 and Analysis of Its Interactions With the Homohexameric AAA + ATPase p97 (Valosin-Containing Protein).

The abundant homohexameric AAA + ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyzes the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterized UBX domain containing protein 9 (UBXD9). Here, we analyzed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum. Pull-down assays revealed that the UBX domain (amino acids 384–466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β0-α–1-α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261–573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261–573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilizing the p97 D1:D1’ intermolecular interface. We thus propose that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers. Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organization of the actin cytoskeleton, and are in line with the hypothesized oligomerization-dependent mechanism of p97 regulation.

Multiple domain interfaces mediate SARM1 autoinhibition

Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD+ and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme. Here we discovered multiple intramolecular and intermolecular domain interfaces required for SARM1 autoinhibition using peptide mapping and cryo-electron microscopy (cryo-EM). We identified a candidate autoinhibitory region by screening a panel of peptides derived from the SARM1 ARM domain, identifying a peptide mediating high-affinity inhibition of the SARM1 NADase. Mutation of residues in full-length SARM1 within the region encompassed by the peptide led to loss of autoinhibition, rendering SARM1 constitutively active and inducing spontaneous NAD+ and axon loss. The cryo-EM structure of SARM1 revealed 1) a compact autoinhibited SARM1 octamer in which the TIR domains are isolated and prevented from oligomerization and enzymatic activation and 2) multiple candidate autoinhibitory interfaces among the domains. Mutational analysis demonstrated that five distinct interfaces are required for autoinhibition, including intramolecular and intermolecular ARM-SAM interfaces, an intermolecular ARM-ARM interface, and two ARM-TIR interfaces formed between a single TIR and two distinct ARM domains. These autoinhibitory regions are not redundant, as point mutants in each led to constitutively active SARM1. These studies define the structural basis for SARM1 autoinhibition and may enable the development of SARM1 inhibitors that stabilize the autoinhibited state.

Phosphorylation of RIAM by src promotes integrin activation by unmasking the PH domain of RIAM

Integrin activation controls cell adhesion, migration, invasion, and extracellular matrix remodeling. RIAM (RAP1-GTP-interacting adaptor molecule) is recruited by activated RAP1 to the plasma membrane (PM) to mediate integrin activation via an inside-out signaling pathway. This process requires the association ofthe pleckstrin homology (PH) domain of RIAM with the membrane PIP2. We identify a conserved intermolecular interface that masks the PIP2-binding site in the PH domains of RIAM. Our data indicate that phosphorylation of RIAM by Src family kinases disrupts this PH-mediated interface, unmasks the membrane PIP2-binding site, and promotes integrin activation. We further demonstrate that this process requires phosphorylation of Tyr267 and Tyr427 in the RIAM PH domain by Src. Our data reveal an unorthodox regulatory mechanism of small GTPase effector proteins by phosphorylation-dependent PM association of the PH domain and provide new insights into the link between Src kinases and integrin signaling.

Molecular Interaction Mechanism of a 14-3-3 Protein with a Phosphorylated Peptide Elucidated by Enhanced Conformational Sampling.

Enhanced conformational sampling, a genetic-algorithm-guided multidimensional virtual-system coupled molecular dynamics, can provide equilibrated conformational distributions of a receptor protein and a flexible ligand at room temperature. The distributions provide not only the most stable but also semistable complex structures and propose a ligand-receptor binding process. This method was applied to a system consisting of a receptor protein, 14-3-3ε, and a flexible peptide, phosphorylated myeloid leukemia factor 1 (pMLF1). The results present comprehensive binding pathways of pMLF1 to 14-3-3ε. We identified four thermodynamically stable clusters of MLF1 on the 14-3-3ε surface and free-energy barriers among some clusters. The most stable cluster includes two high-density spots connected by a narrow corridor. When pMLF1 passes the corridor, a salt-bridge relay (switching) related to the phosphorylated residue of pMLF1 occurs. Conformations in one high-density spot are similar to the experimentally determined complex structure. Three-dimensional distributions of residues in the intermolecular interface rationally explain the binding constant changes resulting from the alanine mutation experiment for the residues. We also performed a simulation of nonphosphorylated peptide and 14-3-3ε, which demonstrated that the complex structure was unstable, suggesting that phosphorylation of the peptide is crucially important for binding to 14-3-3ε.

Characterization of Dimeric Interactions within Protrusion-Domain Interfaces of Parallel and X-Shaped Conformations of Macrobrachium rosenbergii Nodavirus: A Theoretical Study Using the DFT Method along with QTAIM and NBO Analyses.

The protrusion domain (P-domain; MrNVPd) of Macrobrachium rosenbergii nodavirus (MrNV) exists in two conformations, parallel and X-shaped. We have performed a theoretical study to gain insight into the nature of the dimeric interactions involving the dimeric interfaces within parallel and X-shaped conformations of MrNVPd by applying the quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses in the framework of the density functional theory (DFT) approach. The results reveal that the dimer–dimer interfaces of MrNVPd have hydrogen bonds of common types. Leu255–Lys287, Tyr257–Lys287, Lys287–Ser253, Met294–Cys328, Asp295–Lys327, Ser298–Ser324, Ile326–Asp295, and Cys328–Met294 are the key residue pairs of the dimer–dimer interfaces to maintain the dimer–dimer structures of MrNVPd through charge–charge, charge–dipole, dipole–dipole, hydrophobic, and hydrogen bonding interactions. The strengths of these intermolecular dimer–dimer interactions in the parallel conformation are much greater than those in the X-shaped conformation. The parallel trimeric interface is held basically by electrostatic and hydrophobic interactions. The electrostatic interactions accompanying a strong hydrogen bond of Oγ1–Hγ1···Oγ1 in the Thr276 A–Thr276 D pair maintain the intermolecular interface of two X-shaped MrNVPd dimers.

Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding.

Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB-DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB-DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

Characterization of H/D exchange in type 1 pili by proton-detected solid-state NMR and molecular dynamics simulations

Uropathogenic Escherichia coli invades and colonizes hosts by attaching to cells using adhesive pili on the bacterial surface. Although many biophysical techniques have been used to study the structure and mechanical properties of pili, many important details are still unknown. Here we use proton-detected solid-state NMR experiments to investigate solvent accessibility and structural dynamics. Deuterium back-exchange at labile sites of the perdeuterated, fully proton back-exchanged pili was conducted to investigate hydrogen/deuterium (H/D) exchange patterns of backbone amide protons in pre-assembled pili. We found distinct H/D exchange patterns in lateral and axial intermolecular interfaces in pili. Amide protons protected from H/D exchange in pili are mainly located in the core region of the monomeric subunit and in the lateral intermolecular interface, whereas the axial intermolecular interface and the exterior region of pili are highly exposed to H/D exchange. Additionally, we performed molecular dynamics simulations of the type 1 pilus rod and estimated the probability of H/D exchange based on hydrogen bond dynamics. The comparison of the experimental observables and simulation data provides insights into stability and mechanical properties of pili.

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding*[S

Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

Investigations into the Structure and Intermolecular Interface of Human Cofilin-2 Assembled on Actin Filaments by Magic Angle Spinning NMR

Actin comprises one major component of the eukaryotic cytoskeleton and is one of the most ubiquitous and conserved proteins. The cofilin/ADF family of proteins play an integral role in the regulation of actin polymerization dynamics. Specifically, by stimulating severing and depolymerization of actin filaments they potentiate actin recycling in a nucleotide-dependent manner. While actin treadmilling is crucial for cell function and is responsible for up to 50% of cellular ATP consumption, there is a lack of atomic-level information regarding the structure and dynamics of cofilin/ADF proteins bound to actin filaments. We have previously reported structural characterization of cofilin/actin assemblies using magic angle spinning (MAS) NMR spectroscopy. Our MAS NMR results for the F-actin binding site agree with the 3.7 Å cryo-EM structure of cofilin/actin complex, with three patches identified on cofilin for F-actin binding. Other structural changes of cofilin that occur from binding to F-actin will be clarified upon the determination of an atomic-resolution structure. Herein, we report investigations into the structure and intermolecular interface of cofilin bound to F-actin in the ADP and ADP-BeFx nucleotide states. We report 13C and 15N resonance assignments for cofilin-2 using homo- and heteronuclear MAS NMR experiments acquired at 19.9 T. We also present 13C-13C correlation spectra using 2-13C and 1,6-13C-labeled cofilin for determination of distance restraints. Paramagnetic relaxation enhancements (PREs) were also used for long-range distances and the cofilin-actin intermolecular interface. We observe several peaks disappear and decrease in intensity. Peak intensities are correlated to proximity to unpaired spins, and are used to derive atomic distances. These are used in conjunction with our resonance assignments and distance restraints from sparse-labeled cofilin to derive the MAS NMR structure of cofilin/F-actin assemblies, which is currently underway.