Protein Interaction Surfaces Research Articles

The TPR-containing domain within Est1 homologs exhibits species-specific roles in telomerase interaction and telomere length homeostasis

BackgroundThe first telomerase-associated protein (Est1) was isolated in yeast due to its essential role in telomere maintenance. The human counterparts EST1A, EST1B, and EST1C perform diverse functions in nonsense-mediated mRNA decay (NMD), telomere length homeostasis, and telomere transcription. Although Est1 and EST1A/B interact with the catalytic subunit of yeast and human telomerase (Est2 and TERT, respectively), the molecular determinants of these interactions have not been elaborated fully.ResultsTo investigate the functional conservation of the EST1 protein family, we performed protein-protein interaction mapping and structure-function analysis. The domain in hEST1A most conserved between species, containing a TPR (tricotetrapeptide repeat), was sufficient for interaction of hEST1A with multiple fragments of hTERT including the N-terminus. Two mutations within the hTERT N-terminus that perturb in vivo function (NAAIRS92, NAAIRS122) did not affect this protein interaction. ScEst1 hybrids containing the TPR of hEST1A, hEST1B, or hEST1C were expressed in yeast strains lacking EST1, yet they failed to complement senescence. Point mutations within and outside the cognate ScEst1 TPR, chosen to disrupt a putative protein interaction surface, resulted in telomere lengthening or shortening without affecting recruitment to telomeres.ConclusionsThese results identify a domain encompassing the TPR of hEST1A as an hTERT interaction module. The TPR of S. cerevisiae Est1 is required for telomerase-mediated telomere length maintenance in a manner that appears separable from telomere recruitment. Discrete residues in or adjacent to the TPR of Est1 also regulate telomere length homeostasis.

Thermodynamic Properties of Water Molecules at a Protein–Protein Interaction Surface

Protein–protein interactions (PPIs) have been identified as a vital regulator of cellular pathways and networks. However, the determinants that control binding affinity and specificity at protein surfaces are incompletely characterized and thus unable to be exploited for the purpose of developing PPI inhibitors to control cellular pathways in disease states. One of the key factors in intermolecular interactions that remains poorly understood is the role of water molecules and in particular the importance of solvent entropy. This factor is expected to be particularly important at protein surfaces, and the release of water molecules from hydrophobic regions is one of the most important drivers of PPIs. In this work, we have studied the protein surface of a mutant of the protein RadA to quantify the thermodynamics of surface water molecules. RadA and its human homologue RAD51 function as recombinases in the process of homologous recombination. RadA binds to itself to form oligomeric structures and thus contains a well-characterized protein–protein binding surface. Similarly, RAD51 binds either to itself to form oligomers or to the protein BRCA2 to form filaments. X-ray crystallography has determined that the same interface functions in both interactions. Work in our group has generated a partially humanized mutant of RadA, termed MAYM, which has been crystallized in the apo form. We studied this apo form of MAYM using a combination of molecular dynamics (MD) simulations and inhomogeneous fluid solvation theory (IFST). The method locates a number of the hydration sites observed in the crystal structure and locates hydrophobic sites where hydrophobic species are known to bind experimentally. The simulations also highlight the importance of the restraints placed on the protein in determining the results. Finally, the results identify a correlation between the predicted entropy of water molecules at a given site and the solvent-accessible surface area and suggest that correlations between water molecules only need to be considered for water molecules separated by less than 3.2 Å. The combination of MD and IFST has been used previously to study PPIs and represents one of the few existing methods to quantify solvent thermodynamics. This is a vital aspect of molecular recognition and one which we believe must be developed.

Type 5 G Protein β Subunit (Gβ5) Controls the Interaction of Regulator of G Protein Signaling 9 (RGS9) with Membrane Anchors

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gβ5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gβ5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gβ5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.

Molecular Design, Functional Characterization and Structural Basis of a Protein Inhibitor Against the HIV-1 Pathogenicity Factor Nef

Increased spread of HIV-1 and rapid emergence of drug resistance warrants development of novel antiviral strategies. Nef, a critical viral pathogenicity factor that interacts with host cell factors but lacks enzymatic activity, is not targeted by current antiviral measures. Here we inhibit Nef function by simultaneously blocking several highly conserved protein interaction surfaces. This strategy, referred to as “wrapping Nef”, is based on structure-function analyses that led to the identification of four target sites: (i) SH3 domain interaction, (ii) interference with protein transport processes, (iii) CD4 binding and (iv) targeting to lipid membranes. Screening combinations of Nef-interacting domains, we developed a series of small Nef interacting proteins (NIs) composed of an SH3 domain optimized for binding to Nef, fused to a sequence motif of the CD4 cytoplasmic tail and combined with a prenylation signal for membrane association. NIs bind to Nef in the low nM affinity range, associate with Nef in human cells and specifically interfere with key biological activities of Nef. Structure determination of the Nef-inhibitor complex reveals the molecular basis for binding specificity. These results establish Nef-NI interfaces as promising leads for the development of potent Nef inhibitors.

Mapping the protein-protein interface between a toxin and its cognate antitoxin from the bacterial pathogen Streptococcus pyogenes.

Protein--protein interactions are ubiquitous and essential for most biological processes. Although new proteomic technologies have generated large catalogs of interacting proteins, considerably less is known about these interactions at the molecular level, information that would aid in predicting protein interactions, designing therapeutics to alter these interactions, and understanding the effects of disease-producing mutations. Here we describe mapping the interacting surfaces of the bacterial toxin SPN (Streptococcus pyogenes NAD(+) hydrolase) in complex with its antitoxin IFS (immunity factor for SPN) by using hydrogen-deuterium amide exchange and electrospray ionization mass spectrometry. This approach affords data in a relatively short time for small amounts of protein, typically 5-7 pmol per analysis. The results show a good correspondence with a recently determined crystal structure of the IFS--SPN complex but additionally provide strong evidence for a folding transition of the IFS protein that accompanies its binding to SPN. The outcome shows that mass-based chemical footprinting of protein interaction surfaces can provide information about protein dynamics that is not easily obtained by other methods and can potentially be applied to large, multiprotein complexes that are out of range for most solution-based methods of biophysical analysis.

Structural Biology by Mass Spectrometry: Mapping Protein Interaction Surfaces of Membrane Receptor Complexes with ICAT

Multi-component protein complexes underlie most high-order cellular functions. Elucidating the structures of these complexes and understanding how the interactions that bind them change under perturbation is a major goal of structural biology. To help in this effort, Underbakke et al provide a new twist on the proteomics technique of isotope coded affinity tagging (ICAT). In proteomics, ICAT allows relative quantification of proteins in comparable samples by reacting their exposed cysteine thiols with alkylating agents that contain either a heavy (13C) or light (12C) isotope and an affinity tag1; 2. One sample/condition is modified with the heavy tag, the other with the light, they are mixed, proteolytically cleaved into peptides, affinity purified and analyzed by mass spectrometry. Relative signals from the two mass tags report on the availability of the cysteine for modification in the two conditions. This availability reflects how much of a given cysteine residue (and its cognate protein) is there to be labeled, or in the present example, how reactive the cysteine residue is under the condition tested. Originally, the tag employed was a biotin moiety1, but this has been adapted to smaller, glucanime-based labels that can be enriched on a boronate matrix3; 4. Silverman and Harbury applied this method to the foot printing of protein accessibility and interaction surfaces4, in a manner that parallels the logic of hydrogen exchange mass spectrometery5. The current report was predicated by the author’s development of new ICAT reagents based on the glucamine backbone that are more water soluble, but have varying degrees of electrophilicity6. By measuring rates of alklyation (not just total amounts of product), the relative reactivity of cysteine residues that were specifically targeted to a protein surface could be better quantified. Here, the authors extend these studies to mapping interactions within the receptor kinase complexes that compose the bacterial chemotaxis transmembrane signaling system. Bacterial chemotaxis relys on polar clusters of transmembrane chemoreceptors coupled to the histidine kinase CheA through an adaptor protein CheW. There is great interest in the detailed architecture of the receptor arrays and how CheA activity is regulated by ligands7. Biochemical protection assays8; 9, genetic studies10; 11; 12; 13 and structural work14; 15 have probed functional interfaces in this ultrastable complex16, making it an ideal subject for testing new mapping techniques. The authors target the CheW coupling protein because it interacts with both CheA and the receptors. By substituting surface residues for cysteine and evaluating ICAT reactivity in the absence and presence of the other components they identify CheA and receptor binding regions of CheW that are largely consistent with the previous studies mentioned above. Unlike the proteomics application, careful work must be done to target the Cys residues appropriately and verify that substitution and subsequent modification does not alter activity; however, the affinity purification is a big advantage in avoiding background labeling in the complex mixture of native bacterial membranes. Importantly, the resolution of the technique is at the residue level, and has the potential to reveal how interactions between the components change subtly as they switch among activity states.

Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.

BackgroundThe direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.Methodology/Principal FindingsWe report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.Conclusions/SignificanceGenetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

Crystal Structures of Human Exonuclease I with DNA Provides Insight into Substrate Selectivity and Mechanism

Human Exonuclease 1 (hEXO1) plays an essential role in the maintenance of genomic stability through nuclease activity on DNA intermediates involved in replication, recombination, and repair. hEXO1 is a member of the structure-specific 5’ nuclease family, which includes FEN-1 (flap endonuclease 1), GEN-1 (gap endonulcease 1), and XPG (xeroderma pigmentosum complementation group G). This family possesses highly divergent protein sequences and DNA substrate specificities. We present crystal structures of the hEXOI catalytic domain in complex with a 5’ recessed DNA substrate, which represents an intermediate structure in mismatch repair. The structure contains a core region seen in a variety of homologous FEN-1 structures; however, certain nucleotide binding motifs are altered or repositioned. These structures with DNA bound in an assembled active site provide the insight into the mechanism of EXO1 and important family members. Contacts to the DNA are made primarily to the complementary strand, anchored by a potassium-binding motif observed previously in Pol β. Additional contacts are made through a hydrophobic wedge which induces a ∼90° bend in the DNA. The 5’ substrate strand is held in place mainly by base pairing; however, two bases must unpair from the substrate strand to reach the active site. In a product complex the terminal base in the active site is flipped and stabilized by pi-stacking interactions with a tyrosine. The terminal phosphate is coordinated by two Mn2+ ions in a geometry consistent with other nucleases that cleave DNA using a two-metal ion mechanism for catalysis. Structures of hEXO1 will greatly enhance understanding of substrate selection mechanisms and nuclease family mode of action, as well as allowing us to model additional substrates. Analysis of these structures may permit identification of protein-interaction surfaces critical to DNA repair.

Inducible SUMO modification of TANK alleviates its repression of TLR7 signalling

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.

The Crystal Structure of the Dachshund Domain of Human SnoN Reveals Flexibility in the Putative Protein Interaction Surface

The human SnoN is an oncoprotein that interacts with several transcription-regulatory proteins such as the histone-deacetylase, N-CoR containing co-repressor complex and Smad proteins. This study presents the crystal structure of the Dachshund homology domain of human SnoN. The structure reveals a groove composed of conserved residues with characteristic properties of a protein-interaction surface. A comparison of the 12 monomers in the asymmetric unit reveals the presence of two major conformations: an open conformation with a well accessible groove and a tight conformation with a less accessible groove. The variability in the backbone between the open and the tight conformations matches the differences seen in previously determined structures of individual Dachshund homology domains, suggesting a general plasticity within this fold family. The flexibility observed in the putative protein binding groove may enable SnoN to recognize multiple interaction partners.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Insulation of a synthetic hydrogen metabolism circuit in bacteria

BackgroundThe engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued.ResultsHere we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds.ConclusionsThrough the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.

Arginine/lysine–methyl/methyl switches: biochemical role of histone arginine methylation in transcriptional regulation

Post-translational modifications (PTMs) are commonly used to modify protein function. Modifications such as phosphorylation, acetylation and methylation can influence the conformation of the modified protein and its interaction with other proteins or DNA. In the case of histones, PTMs on specific residues can influence chromatin structure and function by modifying the biochemical properties of key amino acids. Histone methylation events, especially on arginine- and lysine-residues, are among the best-characterized PTMs, and many of these modifications have been linked to downstream effects. The addition of a methyl group to either residue results in a slight increase in hydrophobicity, in the loss of a potential hydrogen-bond donor site and, in the alteration of the protein interaction surface. Thus far, a number of protein domains have been demonstrated to directly bind to methylated lysine residues. However, the biochemical mechanisms linking histone arginine methylation to downstream biological outputs remain poorly characterized. This review will focus on the role of histone arginine methylation in transcriptional regulation and on the crosstalk between arginine methylation and other PTMs. We will discuss the mechanisms by which differentially methylated arginines on histones modulate transcriptional outcomes and contribute to the complexity of the 'histone code'.

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein–protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP+ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins.

Drosophila expresses a CD98 transporter with an evolutionarily conserved structure and amino acid-transport properties

Mammalian CD98 heterodimeric amino acid transporters consist of a promiscuous single-pass transmembrane glycoprotein, CD98hc (CD98 heavy chain), and one of six multipass transmembrane proteins or 'light chains'. The heterodimeric complexes of CD98hc and the light chains LAT1 (L-type amino acid transporter 1) or LAT2 specifically promote sodium-independent System L exchange of neutral amino acids, including leucine. CD98hc is also implicated in other processes, including cell fusion, cell adhesion and activation of TOR (target of rapamycin) signalling. Surprisingly, recent reports suggested that insects lack a membrane-bound CD98hc, but in the present study we show that Drosophila CG2791 encodes a functional CD98hc orthologue with conservation in intracellular, transmembrane and extracellular domains. We demonstrate by RNA-interference knockdown in Drosophila Schneider cells that CG2791 and two Drosophila homologues of the mammalian CD98 light chains, Mnd (Minidiscs) and JhI-21, are required for normal levels of System L transport. Furthermore, we show that System L activity is increased by methoprene, an analogue of the developmentally regulated endocrine hormone juvenile hormone, an effect that is potentially mediated by elevated Mnd expression. Co-expression of CG2791 and JhI-21, but not CG2791 and Mnd, in Xenopus oocytes mediates System L transport. Finally, mapping of conserved sequences on to the recently determined crystal structure of the human CD98hc extracellular domain highlights two conserved exposed hydrophobic patches at either end of the domain that are potential protein-protein-interaction surfaces. Therefore our results not only show that there is functional conservation of CD98hc System L transporters in flies, but also provide new insights into the structure, functions and regulation of heterodimeric amino acid transporters.

Highly automated protein backbone resonance assignment within a few hours: the «BATCH» strategy and software package

Sequential resonance assignment represents an essential step towards the investigation of protein structure, dynamics, and interaction surfaces. Although the experimental sensitivity has significantly increased in recent years, with the availability of high field magnets and cryogenically cooled probes, resonance assignment, even of small globular proteins, still generally requires several days of data collection and analysis using standard protocols. Here we introduce the BATCH strategy for fast and highly automated backbone resonance assignment of (13)C, (15)N-labelled proteins. BATCH makes use of the fast data acquisition and analysis tools BEST, ASCOM, COBRA, and HADAMAC, recently developed in our laboratory. An improved Hadamard encoding scheme, presented here, further increases the performance of the HADAMAC experiment. A new software platform, interfaced to the NMRView software package, has been developed that enables highly automated NMR data processing and analysis, sequential resonance assignment, and (13)C chemical shift extraction. We demonstrate for four small globular proteins that sequential resonance assignment can be routinely obtained within a few hours, or less, in a highly automated and robust way.

Differential Presentation of Protein Interaction Surfaces on the Androgen Receptor Defines the Pharmacological Actions of Bound Ligands

The pharmacological activity of different nuclear receptor ligands is reflected by their impact on receptor structure. Thus, we asked whether differential presentation of protein-protein interaction surfaces on the androgen receptor (AR), a surrogate assay of receptor conformation, could be used in a prospective manner to define the pharmacological activity of bound ligands. To this end, we identified over 150 proteins/polypeptides whose ability to interact with AR is influenced in a differential manner by ligand binding. The most discriminatory of these protein-AR interactions were used to develop a robust compound-profiling tool that enabled the separation of ligands into functionally distinguishable classes. Importantly, the ligands within each class exhibited similar pharmacological activities, a result that highlights the relationship between receptor structure and activity and provides direction for the discovery of novel AR modulators.

An Efficient Strategy for the Determination of the Three-Dimensional Architecture of Ribonucleoprotein Complexes by the Combination of a Few Easily Accessible NMR and Biochemical Data: Intermolecular Recognition in a U4 Spliceosomal Complex

Ribonucleoprotein (RNP) complexes are involved in several cellular processes, including RNA processing, transcription and translation. RNP structures are often dynamic in nature, undergoing significant remodeling during the course of their function. Visualization of the three-dimensional arrangement of single components in the complex and characterization of the intermolecular interactions are essential for understanding the mechanisms of operation. Crystallization either is not always achievable for these highly dynamic RNP particles or requires trimming the complex to a stable, well-structured core that lacks the flexible, regulatory domains. Alternative techniques that can provide structural information for complexes in solution under native conditions, where they retain their natural dynamic properties, are needed. In this study, we explored the possibility of using a combination of NMR, biochemical data and molecular modeling to generate an accurate high-resolution model of RNP complexes. We applied this strategy to the ternary hPrp31 (human Prp31)-15.5K-U4 5'-SL (stem-loop) spliceosomal complex, which, due to its large size and instability and because of the difficulty in obtaining isotopically labeled hPrp31, is not amenable to complete structure determination by NMR. We designed a protocol where the protein-protein interaction surface is defined for 15.5K by NMR data, while the relative orientations of the U4 RNA and the hPrp31 protein are described by mutational and cross-linking data. Using these data in a restrained ensemble docking protocol, we obtained a model for the ternary complex that reveals a novel rationale for the hierarchical assembly of the complex. Comparison of the docking model with the crystal structure recently obtained for a trimmed version of the complex reveals the high accuracy of the docking model, even down to an atomic level. This work shows that the architecture of large RNP complexes is within reach by NMR investigation in solution even for those cases where a traditional structural determination cannot be performed.

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 General Method for Scanning Unnatural Amino Acid Mutagenesis

Current approaches to protein site-directed mutagenesis require an independent user operation for each mutation. This can impede large-scale scanning mutagenesis projects such as the mapping of protein interaction surfaces, active sites, or epitopes. It also prevents the creation of protein libraries of defined complexity for directed evolution purposes. Here we present a simple, fast, and effective way to perform scanning codon mutagenesis throughout a protein sequence. The process allows the researcher to define the new codon change, and therefore any amino acid mutation can be achieved. We demonstrate this approach by creating a library of proteins that contain single unnatural amino acid mutations encoded by the amber stop codon, TAG. The mutant proteins generated by this method can be expressed and assayed individually or used together as a mixed population of "rationally diversified" protein sequences.