Supramodular nature of GRIP1 revealed by the structure of its PDZ12 tandem in complex with the carboxyl tail of Fras1

The scaffold protein GRIP1 (glutamate receptor interacting protein 1) binds to and regulates both the trafficking and membrane organization of a large number of transmembrane proteins. Mutation of GRIP1 in mice displays essentially the same phenotype of the mutations of Fras1 or Frem2, which are the animal models of the human genetic disorder Fraser syndrome. However, the molecular basis governing the interaction between GRIP1 and Fras1/Frem2 is unknown. Here, we show that interaction between Fras1 and GRIP1 requires the first two PDZ domains (PDZ1 and PDZ2) to be connected in tandem, as the folding of PDZ1 strictly depends on the covalent attachment of PDZ2. The crystal structure of GRIP1 PDZ12 in complex with the Fras1 C-terminal peptide reveals that the PDZ12 tandem forms a supramodule in which only the peptide-binding groove of PDZ1 is bound with the Fras1 peptide. The GRIP1 PDZ12/Fras1 peptide complex not only provides a mechanistic explanation of the link between GRIP1 and the Fraser syndrome but may also serve as a foundation for searching for potential mutations in GRIP1 that could lead to the Fraser syndrome.     

Structure, Dynamics and Folding of an Immunoglobulin Domain of the Gelation Factor (ABP-120) from Dictyostelium discoideum

We have carried out a detailed structural and dynamical characterisation of the isolated fifth repeat of the gelation factor (ABP-120) from Dictyostelium discoideum (ddFLN5) by NMR spectroscopy to provide a basis for studies of co-translational folding on the ribosome of this immunoglobulin-like domain. The isolated ddFLN5 can fold autonomously in solution into a structure that resembles very closely the crystal structure of the domain in a construct in which the adjacent sixth repeat (ddFLN6) is covalently linked to its C-terminus in tandem but deviates locally from a second crystal structure in which ddFLN5 is flanked by ddFLN4 and ddFLN6 at both N- and C-termini. Conformational fluctuations were observed via ¹⁵N relaxation methods and are primarily localised in the interstrand loops that encompass the C-terminal hemisphere. These fluctuations are distinct in location from the region where line broadening is observed in ddFLN5 when attached to the ribosome as part of a nascent chain. This observation supports the conclusion that the broadening is associated with interactions with the ribosome surface [Hsu, S. T. D., Fucini, P., Cabrita, L. D., Launay, H., Dobson, C. M. & Christodoulou, J. (2007). Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy. Proc. Natl. Acad. Sci. USA, 104, 16516-16521]. The unfolding of ddFLN5 induced by high concentrations of urea shows a low population of a folding intermediate, as inferred from an intensity-based analysis, a finding that differs from that of ddFLN5 as a ribosome-bound nascent chain. These results suggest that interesting differences in detail may exist between the structure of the domain in isolation and when linked to the ribosome and between protein folding in vitro and the folding of a nascent chain as it emerges from the ribosome.     

Design and Structure of an Equilibrium Protein Folding Intermediate: A Hint into Dynamical Regions of Proteins

Partly unfolded protein conformations close to the native state may play important roles in protein function and in protein misfolding. Structural analyses of such conformations which are essential for their fully physicochemical understanding are complicated by their characteristic low populations at equilibrium. We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR. It consists of a large native region identical with that observed in the X-ray structure of the wild-type protein plus an unfolded region. Small-angle X-ray scattering analysis indicates that the calculated ensemble of structures is consistent with the actual degree of expansion of the intermediate. The unfolded region encompasses discontinuous sequence segments that cluster in the 3D structure of the native protein forming the FMN cofactor binding loops and the binding site of a variety of partner proteins. Analysis of the apoflavodoxin inner interfaces reveals that those becoming destabilized in the intermediate are more polar than other inner interfaces of the protein. Natively folded proteins contain hydrophobic cores formed by the packing of hydrophobic surfaces, while natively unfolded proteins are rich in polar residues. The structure of the apoflavodoxin thermal intermediate suggests that the regions of natively folded proteins that are easily responsive to thermal activation may contain cores of intermediate hydrophobicity.     

The crystal structure of the secreted dimeric form of the hemophore HasA reveals a domain swapping with an exchanged heme ligand

To satisfy their iron needs, several Gram-negative bacteria use a heme uptake system involving an extracellular heme-binding protein called hemophore. The function of the hemophore is to acquire free or hemoprotein-bound heme and to transfer it to HasR, its specific outer membrane receptor, by protein-protein interaction. The hemophore HasA secreted by Serratia marcescens, an opportunistic pathogen, was the first to be identified and is now very well characterized. HasA is a monomer that binds one b heme with strong affinity. The heme in HasA is highly exposed to solvent and coordinated by an unusual pair of ligands, a histidine and a tyrosine. Here, we report the identification, the characterization and the X-ray structure of a dimeric form of HasA from S. marcescens: DHasA. We show that both monomeric and dimeric forms are secreted in iron deficient conditions by S. marcescens. The crystal structure of DHasA reveals that it is a domain swapped dimer. The overall structure of each monomeric subunit of DHasA is very similar to that of HasA but formed by parts coming from the two different polypeptide chains, involving one of the heme ligands. Consequently DHasA binds two heme molecules by residues coming from both polypeptide chains. We show here that, while DHasA can bind two heme molecules, it is not able to deliver them to the receptor HasR. However, DHasA can efficiently transfer its heme to the monomeric form that, in turn, delivers it to HasR. We assume that DHasA can function as a heme reservoir in the hemophore system.     

Conformational Dynamics and Structural Plasticity Play Critical Roles in the Ubiquitin Recognition of a UIM Domain

Ubiquitin-interacting motifs (UIMs) are an important class of protein domains that interact with ubiquitin or ubiquitin-like proteins. These approximately 20-residue-long domains are found in a variety of ubiquitin receptor proteins and serve as recognition modules towards intracellular targets, which may be individual ubiquitin subunits or polyubiquitin chains attached to a variety of proteins. Previous structural studies of interactions between UIMs and ubiquitin have shown that UIMs adopt an extended structure of a single α-helix, containing a hydrophobic surface with a conserved sequence pattern that interacts with key hydrophobic residues on ubiquitin. In light of this large body of structural studies, details regarding the presence and the roles of structural dynamics and plasticity are surprisingly lacking. In order to better understand the structural basis of ubiquitin-UIM recognition, we have characterized changes in the structure and dynamics of ubiquitin upon binding of a UIM domain from the yeast Vps27 protein. The solution structure of a ubiquitin-UIM fusion protein designed to study these interactions is reported here and found to consist of a well-defined ubiquitin core and a bipartite UIM helix. Moreover, we have studied the plasticity of the docking interface, as well as global changes in ubiquitin due to UIM binding at the picoseconds-to-nanoseconds and microseconds-to-milliseconds protein motions by nuclear magnetic resonance relaxation. Changes in generalized-order parameters of amide groups show a distinct trend towards increased structural rigidity at the UIM-ubiquitin interface relative to values determined in unbound ubiquitin. Analysis of ¹⁵N Carr-Purcell-Meiboom-Gill relaxation dispersion measurements suggests the presence of two types of motions: one directly related to the UIM-binding interface and the other induced to distal parts of the protein. This study demonstrates a case where localized interactions among protein domains have global effects on protein motions at timescales ranging from picoseconds to milliseconds.     

One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase

Acireductone dioxygenase (ARD) catalyzes different reactions between O2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni2+ -ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe2+ is bound (ARD'), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe2+ renders the active site of ARD' inaccessible to standard NMR methods. The structure of ARD' has been determined using Fe2+ binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD'. ARD' retains the beta-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the beta-sandwich and decreases order at the other upon interconversion of ARD and ARD', causing loss of the C-terminal helix in ARD' and rearrangements of residues involved in substrate orientation in the active site.     

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

In the N2 domain of the gene-3-protein of phage fd, two consecutive β-strands are connected by a mobile loop of seven residues (157-163). The stability of this loop is low, and the Asp160-Pro161 bond at its tip shows conformational heterogeneity with 90% being in the cis and 10% in the trans form. The refolding kinetics of N2 are complex because the molecules with cis or trans isomers at Pro161 both fold to native-like conformations, albeit with different rates. We employed consensus design to shorten the seven-residue irregular loop around Pro161 to a four-residue type I′ turn without a proline. This increased the conformational stability of N2 by almost 10 kJ mol^− 1 and abolished the complexity of the folding kinetics. Turn sequences obtained from in vitro selections for increased stability strongly resembled those derived from the consensus design. Two other type I′ turns of N2 could also be stabilized by consensus design. For all three turns, the gain in stability originates from an increase in the rate of refolding. The turns form native-like structures early during refolding and thus stabilize the folding transition state. The crystal structure of the variant with all three stabilized turns confirms that the 157-163 loop was in fact shortened to a type I′ turn and that the other turns maintained their type I′ conformation after sequence optimization.     

Structural and Motional Contributions of the Bacillus subtilis ClpC N-Domain to Adaptor Protein Interactions

The AAA ⁺ (ATPases associated with a variety of cellular activities) superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities. The NMR solution structure of the N-terminal repeat domain of ClpC (N-ClpCR) comprises two structural repeats of a four-helix motif. NMR experiments used to map the MecA adaptor protein interaction surface of N-ClpCR reveal that regions involved in the interaction possess conformational flexibility and conformational exchange on the microsecond-to-millisecond timescale. The electrostatic surface of N-ClpCR differs substantially from the N-domain of Escherichia coli ClpA and ClpB, suggesting that the electrostatic surface characteristics of HSP100/Clp N-domains may play a role in adaptor protein and substrate interaction specificity, and perhaps contribute to the unique adaptor protein requirement of ClpC.     

A Nuclear Localization Signal at the SAM-SAM Domain Interface of AIDA-1 Suggests a Requirement for Domain Uncoupling Prior to Nuclear Import

The neuronal scaffolding protein AIDA-1 is believed to act as a convener of signals arising at postsynaptic densities. Among the readily identifiable domains in AIDA-1, two closely juxtaposed sterile alpha motif (SAM) domains and a phosphotyrosine binding domain are located within the C-terminus of the longest splice variant and exclusively in four shorter splice variants. As a first step towards understanding the possible emergent properties arising from this assembly of ligand binding domains, we have used NMR methods to solve the first structure of a SAM domain tandem. Separated by a 15-aa linker, the two SAM domains are fused in a head-to-tail orientation that has been observed in other hetero- and homotypic SAM domain structures. The basic nuclear import signal for AIDA-1 is buried at the interface between the two SAM domains. An observed disparity between the thermal stabilities of the two SAM domains suggests a mechanism whereby the second SAM domain decouples from the first SAM domain to facilitate translocation of AIDA-1 to the nucleus.     

The solution structure of the C-terminal modular pair from Clostridium perfringens mu-toxin reveals a noncellulosomal dockerin module

The genome of the opportunistic pathogen Clostridium perfringens encodes a large number of secreted glycoside hydrolases. Their predicted activities indicate that they are involved in the breakdown of complex carbohydrates and other glycans found in the mucosal layer of the human gastrointestinal tract, within the extracellular matrix, and on the surface of host cells. One such group of these enzymes is the family 84 glycoside hydrolases, which has predicted hyaluronidase activity and comprises five members [C. perfringens glycoside hydrolase family 84 (CpGH84) A-E]. The first identified member, CpGH84A, corresponds to the μ-toxin whose modular architecture includes an N-terminal catalytic domain, four family 32 carbohydrate-binding modules, three FIVAR modules of unknown function, and a C-terminal putative calcium-binding module. Here, we report the solution NMR structure of the C-terminal modular pair from the μ-toxin. The three-helix bundle FIVAR module displays structural homology to a heparin-binding module within the N-terminal of the a C protein from group B Streptoccocus. The C-terminal module has a typical calcium-binding dockerin fold comprising two anti-parallel helices that form a planar face with EF-hand calcium-binding loops at opposite ends of the module. The size of the helical face of the μ-toxin dockerin module is approximately equal to the planar region recently identified on the surface of a cohesin-like X82 module of CpGH84C. Size-exclusion chromatography and heteronuclear NMR-based chemical shift mapping studies indicate that the helical face of the dockerin module recognizes the CpGH84C X82 module. These studies represent the structural characterization of a noncellulolytic dockerin module and its interaction with a cohesin-like X82 module. Dockerin/X82-mediated enzyme complexes may have important implications in the pathogenic properties of C. perfringens.     

SAXS and X-ray Crystallography Suggest an Unfolding Model for the GDP/GTP Conformational Switch of the Small GTPase Arf6

The small GTPases Arf1 and Arf6 have nonoverlapping functions in cellular traffic despite their very high sequence and structural resemblance. Notably, the exquisite isoform specificity of their guanine nucleotide exchange factors and their distinctive sensitivity to the drug brefeldin A cannot be explained by any straightforward structural model. Here we integrated structural and spectroscopic methods to address this issue using Δ13Arf6-GDP, a truncated mutant that mimics membrane-bound Arf6-GDP. The crystal structure of Δ13Arf6-GDP reveals an unprecedented unfolding of the GTPase core β-strands, which is fully accounted for by small-angle X-ray scattering data in solution and by ab initio three-dimensional envelope calculation. NMR chemical shifts identify this structural disorder in Δ13Arf6-GDP, but not in the closely related Δ17Arf1-GDP, which is consistent with their comparative thermodynamic and hydrodynamic analyses. Taken together, these experiments suggest an unfolding model for the nucleotide switch of Arf6 and shed new light on its biochemical differences with Arf1.     

Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases

Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed α-helical bundle through their N-terminal double GG4-like motif T⁶⁴⁸G⁶⁴⁹X₂G⁶⁵²A⁶⁵³ and glycine zipper motif T⁶⁵²X₃S⁶⁵⁶X₃G⁶⁶⁰, respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.     

Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation

Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF₃ ⁻ addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.     

Solution structure and dynamics of the N-terminal cytosolic domain of rhomboid intramembrane protease from Pseudomonas aeruginosa: insights into a functional role in intramembrane proteolysis

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.     

Structure and Function of Palladin's Actin Binding Domain

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.     

Binding Hot Spot in the Weak Protein Complex of Physiological Redox Partners Yeast Cytochrome c and Cytochrome c Peroxidase

Transient protein interactions mediate many vital cellular processes such as signal transduction or intermolecular electron transfer. However, due to difficulties associated with their structural characterization, little is known about the principles governing recognition and binding in weak transient protein complexes. In particular, it has not been well established whether binding hot spots, which are frequently found in strong static complexes, also govern transient protein interactions. To address this issue, we have investigated an electron transfer complex of physiological partners from yeast: yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP). Using isothermal titration calorimetry and NMR spectroscopy, we show that Cc R13 is a hot-spot residue, as R13A mutation has a strong destabilizing effect on binding. Furthermore, we employ a double-mutant cycle to illustrate that Cc R13 interacts with CcP Y39. The present results, in combination with those of earlier mutational studies, have enabled us to outline the extent of the energetically important Cc-CcP binding region. Based on our analysis, we propose that binding energy hot spots, which are prevalent in static protein complexes, could also govern transient protein interactions.     

Structural Basis for Par-4 Recognition by the SPRY Domain- and SOCS Box-Containing Proteins SPSB1, SPSB2, and SPSB4

The mammalian SPRY domain- and SOCS box-containing proteins, SPSB1 to SPSB4, belong to the SOCS box family of E3 ubiquitin ligases. Substrate recognition sites for the SPRY domain are identified only for human Par-4 (ELNNNL) and for the Drosophila orthologue GUSTAVUS binding to the DEAD-box RNA helicase VASA (DINNNN). To further investigate this consensus motif, we determined the crystal structures of SPSB1, SPSB2, and SPSB4, as well as their binding modes and affinities for both Par-4 and VASA. Mutation of each of the three Asn residues in Par-4 abrogated binding to all three SPSB proteins, while changing EL to DI enhanced binding. By comparison to SPSB1 and SPSB4, the more divergent protein SPSB2 showed only weak binding to Par-4 and was hypersensitive to DI substitution. Par-4_(59-77) binding perturbed NMR resonances from a number of SPSB2 residues flanking the ELNNN binding site, including loop D, which binds the EL/DI sequence. Although interactions with the consensus peptide motif were conserved in all structures, flanking sites in SPSB2 were identified as sites of structural change. These structural changes limit high-affinity interactions for SPSB2 to aspartate-containing sequences, whereas SPSB1 and SPSB4 bind strongly to both Par-4 and VASA peptides.