Deuterium spin relaxation of fractionally deuterated ribonuclease H using paired 475 and 950 MHz NMR spectrometers.

Deuterium (2H) spin relaxation of 13CH2D methyl groups has been widely applied to investigate picosecond-to-nanosecond conformational dynamics in proteins by solution-state NMR spectroscopy. The B0 dependence of the 2H spin relaxation rates is represented by a linear relationship between the spectral density function at three discrete frequencies J(0), J(ωD) and J(2ωD). In this study, the linear relation between 2H relaxation rates at B0 fields separated by a factor of two and the interpolation of rates at intermediate frequencies are combined for a more robust approach for spectral density mapping. The general usefulness of the approach is demonstrated on a fractionally deuterated (55%) and alternate 13C-12C labeled sample of E. coli RNase H. Deuterium relaxation rate constants (R1, R1ρ, RQ, RAP) were measured for 57 well-resolved 13CH2D moieties in RNase H at 1H frequencies of 475 MHz, 500 MHz, 900 MHz, and 950 MHz. The spectral density mapping of the 475/950 MHz data combination was performed independently and jointly to validate the expected relationship between data recorded at B0 fields separated by a factor of two. The final analysis was performed by jointly analyzing 475/950 MHz rates with 700 MHz rates interpolated from 500/900 MHz data to yield six J(ωD) values for each methyl peak. The J(ω) profile for each peak was fit to the original (τM, Sf2, τf) or extended model-free function (τM, Sf2, Ss2, τf, τs) to obtain optimized dynamic parameters.

Dynamics in natural and designed elastins and their relation to elastic fiber structure and recoil.

Elastin fibers assemble in the extracellular matrix from the precursor protein tropoelastin and provide the flexibility and spontaneous recoil required for arterial function. Unlike many proteins, a structure-function mechanism for elastin has been elusive. We have performed detailed NMR relaxation studies of the dynamics of the minielastins 24x' and 20x' using solution NMR, and of purified bovine elastin fibers in the presence and absence of mechanical stress using solid state NMR. The low sequence complexity of the minielastins enables us to determine average dynamical timescales and degrees of local ordering in the cross-link and hydrophobic modules separately using NMR relaxation by taking advantage of their residue-specific resolution. We find an extremely high degree of disorder, with order parameters for the entirety of the hydrophobic domains near zero, resembling that of simple chemical polymers and less than the order parameters that have been observed in other intrinsically disordered proteins. We find that average backbone order parameters in natural, purified elastin fibers are comparable to those found in 24x' and 20x' in solution. The difference in dynamics, compared with the minielastins, is that backbone correlation times are significantly slowed in purified elastin. Moreover, when elastin is mechanically stretched, the high chain disorder in purified elastin is retained, showing that any change in local ordering is below that detectable in our experiment. Combined with our previous finding of a 10-fold increase in the ordering of water when fully hydrated elastin fibers are stretched by 50%, these results support the hypothesis that stretch induced solvent ordering, i.e., the hydrophobic effect, is a key player in the elastic recoil of elastin as opposed to configurational entropy loss.

Dynamic structure of the full-length scaffolding protein NHERF1 influences signaling complex assembly.

The Na+/H+ exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes.

Collagen I Weakly Interacts with the ß-Sheets of ß2-Microglobulin and Enhances Conformational Exchange To Induce Amyloid Formation.

Amyloidogenesis is significant in both protein function and pathology. Amyloid formation of folded, globular proteins is commonly initiated by partial or complete unfolding. However, how this unfolding event is triggered for proteins that are otherwise stable in their native environments is not well understood. The accumulation of the immunoglobulin protein ß2-microglobulin (ß2m) into amyloid plaques in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis (DRA). While ß2m does not form amyloid unassisted near neutral pH in vitro, the localization of ß2m deposits to joint spaces suggests a role for the local extracellular matrix (ECM) proteins, specifically collagens, in promoting amyloid formation. Indeed, collagen and other ECM components have been observed to facilitate ß2m amyloid formation, but the large size and anisotropy of the complex, combined with the low affinity of these interactions, have limited atomic-level elucidation of the amyloid-promoting mechanism(s) by these molecules. Using solution NMR approaches that uniquely probe weak interactions in large molecular weight complexes, we are able to map the binding interfaces on ß2m for collagen I and detect collagen I-induced µs-ms time-scale dynamics in the ß2m backbone. By combining solution NMR relaxation methods and 15N-dark-state exchange saturation transfer experiments, we propose a model in which weak, multimodal collagen I-ß2m interactions promote exchange with a minor population of amyloid-competent species to induce fibrillogenesis. The results portray the intimate role of the environment in switching an innocuous protein into an amyloid-competent state, rationalizing the localization of amyloid deposits in DRA.

Enhanced spectral density mapping through combined multiple-field deuterium 13CH2D methyl spin relaxation NMR spectroscopy.

Quadrupolar relaxation of 2H (D) nuclear spins is a powerful probe of conformational dynamics in biological macromolecules. Deuterium relaxation rate constants are determined by the spectral density function for reorientation of the C-D bond vector at zero, single-quantum, and double-quantum 2H frequencies. In the present work, 2H relaxation rate constants were measured for an E. coli ribonuclease H [U-2H, 15N] ILV-[13CH2D] sample using 400, 500, 800, and 900 MHz NMR spectrometers and analyzed by three approaches to determine spectral density values. First, data recorded at each static magnetic field were analyzed independently. Second, data recorded at 400 and 800 MHz were analyzed jointly and data recorded at other fields were analyzed independently. Third, data recorded at 400 and 500 MHz were interpolated to 450 MHz, and the resulting two pairs of data, corresponding to 400 MHz/800 MHz and 450 MHz/900 MHz, were analyzed jointly. The second and third approaches rely on the identity between the double quantum frequency at the lower field and the single quantum frequency at the higher field. Spectral density values for 32 of the 48 resolvable ILV methyl resonances were fit by the Lipari-Szabo model-free formalism and used to validate the three methods. The three spectral density mapping methods performed equally well in cross validation with data recorded at 700 MHz. However, the third method yielded approximately 10-15% more precise estimates of model-free parameters and consequently provides a general strategy for analysis of 2H spin relaxation data in biological macromolecules.

Correlated motions of C'-N and Cα-Cß pairs in protonated and per-deuterated GB3.

We investigated correlated µs-ms time scale motions of neighboring 13C'-15N and 13Cα-13Cß nuclei in both protonated and perdeuterated samples of GB3. The techniques employed, NMR relaxation due to cross-correlated chemical shift modulations, specifically target concerted changes in the isotropic chemical shifts of the two nuclei associated with spatial fluctuations. Field-dependence of the relaxation rates permits identification of the parameters defining the chemical exchange rate constant under the assumption of a two-site exchange. The time scale of motions falls into the intermediate to fast regime (with respect to the chemical shift time scale, 100-400 s-1 range) for the 13C'-15N pairs and into the slow to intermediate regime for the 13Cα-13Cß pairs (about 150 s-1). Comparison of the results obtained for protonated and deuterated GB3 suggests that deuteration has a tendency to reduce these slow scale correlated motions, especially for the 13Cα-13Cß pairs.

Methyl Relaxation Measurements Reveal Patterns of Fast Dynamics in a Viral RNA-Directed RNA Polymerase.

Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues. Our results, which represent the most detailed experimental characterization of fast dynamics in a viral RdRp until date, reveal a highly connected dynamic network as predicted by MD simulations of related systems. Our results suggest that the entry portals for template RNA and substrate NTPs are relatively disordered, while conserved motifs involved in metal binding, nucleotide selection, and catalysis display greater rigidity. Perturbations at the active site through metal binding or functional mutation affect dynamics not only in the immediate vicinity but also at remote regions. Comparison with the limited experimental and extensive functional and in silico results available for homologous systems suggests conservation of the overall pattern of dynamics in viral RdRps.

Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit.

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA's depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

LC-Photo-CIDNP hyperpolarization of biomolecules bearing a quasi-isolated spin pair: Magnetic-Field dependence via a rapid-shuttling device.

Liquid-state low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) is an emerging technology tailored to enhance the sensitivity of NMR spectroscopy via LED- or laser-mediated optical irradiation. LC-photo-CIDNP is particularly useful to detect solvent-exposed aromatic residues (Trp, Tyr), either in isolation or within polypeptides and proteins. This study investigates the magnetic-field dependence of the LC-photo-CIDNP of Trp-α-13C-ß,ß,2,4,5,6,7-d7, a Trp isotopolog bearing a quasi-isolated 1Hα-13Cαspin pair (QISP). We employed a new rapid-shuttling side-illumination field-cycling device that enables ultra-fast (90-120 ms) vertical movements of NMR samples within the bore of a superconducting magnet. Thus, LC-photo-CIDNP hyperpolarization occurs at low field, while hyperpolarized signals are detected at high field (700 MHz). Resonance lineshapes were excellent, and the effect of several fields (1.18-7.08 T range) on hyperpolarization efficiency could be readily explored. Remarkably, unprecedented LC-photo-CIDNP enhancements ε ≅ 1,200 were obtained at 50 MHz (1.18 T), suggesting exciting avenues to hypersensitive LED-enhanced NMR in liquids at low field.

Arabinose- and xylose-modified analogs of 2',3'-cGAMP act as STING agonists.

Stimulator of interferon genes (STING) agonists are promising candidates for vaccine adjuvants and antitumor immune stimulants. The most potent natural agonist of STING, 2',3'-cyclic GMP-AMP (2',3'-cGAMP), is subject to nuclease-mediated inherent metabolic instability, thereby placing limits on its clinical efficacy. Here, we report on a new class of chemically synthesized sugar-modified analogs of 2',3'-cGAMP containing arabinose and xylose sugar derivatives that bind mouse and human STING alleles with high affinity. The co-crystal structures demonstrate that such analogs act as 2',3'-cGAMP mimetics that induce the "closed" conformation of human STING. These analogs show significant resistance to hydrolysis mediated by ENPP1 and increased stability in human serum, while retaining similar potency as 2',3'-cGAMP at inducing IFN-ß secretion from human THP1 cells. The arabinose- and xylose-modified 2',3'-cGAMP analogs open a new strategy for overcoming the inherent nuclease-mediated vulnerability of natural ribose cyclic nucleotides, with the additional benefit of high translational potential as cancer therapeutics and vaccine adjuvants.

Structural and dynamic studies of the peptidase domain from Clostridium thermocellum PCAT1.

The export of antimicrobial peptides is mediated by diverse mechanisms in bacterial quorum sensing pathways. One such binary system employed by gram-positive bacteria is the PCAT1 ABC transporter coupled to a cysteine protease. The focus of this study is the N-terminal C39 peptidase (PEP) domain from Clostridium thermocellum PCAT1 that processes its natural substrate CtA by cleaving a conserved -GG- motif to separate the cargo from the leader peptide prior to secretion. In this study, we are primarily interested in elucidating the dynamic and structural determinants of CtA binding and how it is coupled to cleavage efficiency in the PCAT1 PEP domain. To this end, we have characterized CtA interactions with PEP domain and PCAT1 transporter in detergent micelles using solution nuclear magnetic resonance spectroscopy. The bound CtA structure revealed the disordered C-terminal cargo peptide is linked by a sterically hindered cleavage site to a helix docked within a hydrophobic cavity in the PEP domain. The wide range of internal motions detected by amide nitrogen (N15 ) relaxation measurements in the free enzyme and substrate-bound complex suggests the binding site is relatively floppy. This flexibility plays a key role in the structural rearrangement necessary to relax steric inhibition in the bound substrate. In conjunction with previously reported PCAT1 structures, we offer fresh insight into the ATP-mediated association between PEP and transmembrane domains as a putative mechanism to optimize peptide cleavage by regulating the width and flexibility of the enzyme active site.

Dynamic regulation of Zn(II) sequestration by calgranulin C.

Calgranulin C performs antimicrobial activity in the human immune response by sequestering Zn(II). This biological function is afforded with the aid of two structurally distinct Ca(II)-binding EF hand motifs, wherein one of which bears an unusual amino acid sequence. Here, we utilize solution state NMR relaxation measurements to investigate the mechanism of Ca(II)-modulated enhancement of Zn(II) sequestration by calgranulin C. Using C13 /N15 CPMG dispersion experiments we have measured pH-dependent major and minor state populations exchanging on micro-to-millisecond timescale. This conformational exchange takes place exclusively in the Ca(II)-bound state and can be mapped to residues located in the EF-I loop and the linker between the tandem EF hands. Molecular dynamics (MD) simulations spanning nano-to-microsecond timescale offer insights into the role of pH-dependent electrostatic interactions in EF-hand dynamics. Our results suggest a pH-regulated dynamic equilibrium of conformations that explore a range of "closed" and partially "open" sidechain configurations within the Zn(II) binding site. We propose a novel mechanism by which Ca(II) binding to a non-canonical EF loop regulates its flexibility and tunes the antimicrobial activity of calgranulin C.

A conformational switch in the scaffolding protein NHERF1 controls autoinhibition and complex formation.

The mammalian Na(+)/H(+) exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.

Antiviral activity of α-helical stapled peptides designed from the HIV-1 capsid dimerization domain.

BACKGROUND: The C-terminal domain (CTD) of HIV-1 capsid (CA), like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs. RESULTS: Due to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC), sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease. CONCLUSIONS: These preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind CA-CTD.

Asterix/Gtsf1 links tRNAs and piRNA silencing of retrotransposons.

The Piwi-interacting RNA (piRNA) pathway safeguards genomic integrity by silencing transposable elements (transposons) in the germline. While Piwi is the central piRNA factor, others including Asterix/Gtsf1 have also been demonstrated to be critical for effective silencing. Here, using enhanced crosslinking and immunoprecipitation (eCLIP) with a custom informatic pipeline, we show that Asterix/Gtsf1 specifically binds tRNAs in cellular contexts. We determined the structure of mouse Gtsf1 by NMR spectroscopy and identified the RNA-binding interface on the protein's first zinc finger, which was corroborated by biochemical analysis as well as cryo-EM structures of Gtsf1 in complex with co-purifying tRNA. Consistent with the known dependence of long terminal repeat (LTR) retrotransposons on tRNA primers, we demonstrate that LTR retrotransposons are, in fact, preferentially de-repressed in Asterix mutants. Together, these findings link Asterix/Gtsf1, tRNAs, and LTR retrotransposon silencing and suggest that Asterix exploits tRNA dependence to identify transposon transcripts and promote piRNA silencing.

Site-Specific Lipidation Enhances IFITM3 Membrane Interactions and Antiviral Activity.

Interferon-induced transmembrane proteins (IFITMs) are S-palmitoylated proteins in vertebrates that restrict a diverse range of viruses. S-palmitoylated IFITM3 in particular engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, how S-palmitoylation modulates the structure and biophysical characteristics of IFITM3 to promote its antiviral activity remains unclear. To investigate how site-specific S-palmitoylation controls IFITM3 antiviral activity, we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of cysteine 72 enhances the antiviral activity of IFITM3 by modulating its conformation and interaction with lipid membranes. Collectively, our results demonstrate that site-specific S-palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.

Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia.

The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

The low-pH unfolded state of the C-terminal domain of the ribosomal protein L9 contains significant secondary structure in the absence of denaturant but is no more compact than the low-pH urea unfolded state.

There is considerable interest in the properties of the unfolded states of proteins, particularly unfolded states which can be populated in the absence of high concentrations of denaturants. Interest in the unfolded state ensemble reflects the fact that it is the starting point for protein folding as well as the reference state for protein stability studies and can be the starting state for pathological aggregation. The unfolded state of the C-terminal domain (residues 58-149) of the ribosomal protein L9 (CTL9) can be populated in the absence of denaturant at low pH. CTL9 is a 92-residue globular alpha, beta protein. The low-pH unfolded state contains more secondary structure than the low-pH urea unfolded state, but it is not a molten globule. Backbone ( (1)H, (13)C, and (15)N) NMR assignments as well as side chain (13)C beta and (1)H beta assignments and (15)N R 2 values were obtained for the pH 2.0 unfolded form of CTL9 and for the urea unfolded state at pH 2.5. Analysis of the deviations of the chemical shifts from random coil values indicates that residues that comprise the two helices in the native state show a clear preference for adopting helical phi and psi angles in the pH 2.0 unfolded state. There is a less pronounced but nevertheless clear tendency for residues 107-124 to preferentially populate helical phi and psi values in the unfolded state. The urea unfolded state has no detectable tendency to populate any type of secondary structure even though it is as compact as the pH 2.0 unfolded state. Comparison of the two unfolded forms of CTL9 provides direct experimental evidence that states which differ significantly in their secondary structure can have identical hydrodynamic properties. This in turn demonstrates that global parameters such as R h or R g are very poor indicators of "random coil" behavior.

Structure of the S100A6 complex with a fragment from the C-terminal domain of Siah-1 interacting protein: a novel mode for S100 protein target recognition.

S100A6 is a member of the S100 subfamily of EF-hand Ca (2+) binding proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP or SIP), a subunit of an SCF-like E3 ubiquitin ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal S100A6 binding region in SIP was mapped to a 31-residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189-219) complex revealed that SIP(189-219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structure-based SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions.