Designing cyclic competence-stimulating peptide (CSP) analogs with pan-group quorum-sensing inhibition activity in Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell-cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.

Insights into the Cross Talk between Effector and Allosteric Lobes of KRAS from Methyl Conformational Dynamics.

KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-µs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.

Conformational flexibility in the enterovirus RNA replication platform.

A presumed RNA cloverleaf (5'CL), located at the 5'-most end of the noncoding region of the enterovirus genome, is the primary established site for initiation of genomic replication. Stem-loop B (SLB) and stem-loop D (SLD), the two largest stem-loops within the 5'CL, serve as recognition sites for protein interactions that are essential for replication. Here we present the solution structure of rhinovirus serotype 14 5'CL using a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering. In the absence of magnesium, the structure adopts an open, somewhat extended conformation. In the presence of magnesium, the structure compacts, bringing SLB and SLD into close contact, a geometry that creates an extensive accessible major groove surface, and permits interaction between the proteins that target each stem-loop.

Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain.

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up-regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

Conformational Switch to a ß-Turn in a Staphylococcal Quorum Sensing Signal Peptide Causes a Dramatic Increase in Potency.

We report the solution-phase structures of native signal peptides and related analogs capable of either strongly agonizing or antagonizing the AgrC quorum sensing (QS) receptor in the emerging pathogen Staphylococcus epidermidis. Chronic S. epidermidis infections are often recalcitrant to traditional therapies due to antibiotic resistance and formation of robust biofilms. The accessory gene regulator (agr) QS system plays an important role in biofilm formation in this opportunistic pathogen, and the binding of an autoinducing peptide (AIP) signal to its cognate transmembrane receptor (AgrC) is responsible for controlling agr. Small molecules or peptides capable of modulating this binding event are of significant interest as probes to investigate both the agr system and QS as a potential antivirulence target. We used NMR spectroscopy to characterize the structures of the three native S. epidermidis AIP signals and five non-native analogs with distinct activity profiles in the AgrC-I receptor from S. epidermidis. These studies revealed a suite of structural motifs critical for ligand activity. Interestingly, a unique ß-turn was present in the macrocycles of the two most potent AgrC-I modulators, in both an agonist and an antagonist, which was distinct from the macrocycle conformation in the less-potent AgrC-I modulators and in the native AIP-I itself. This previously unknown ß-turn provides a structural rationale for these ligands' respective biological activity profiles. Development of analogs to reinforce the ß-turn resulted in our first antagonist with subnanomolar potency in AgrC-I, while analogs designed to contain a disrupted ß-turn were dramatically less potent relative to their parent compounds. Collectively, these studies provide new insights into the AIP:AgrC interactions crucial for QS activation in S. epidermidis and advance the understanding of QS at the molecular level.

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

Structural Characterization of Competence-Stimulating Peptide Analogues Reveals Key Features for ComD1 and ComD2 Receptor Binding in Streptococcus pneumoniae.

Streptococcus pneumoniae is an important pathogen that utilizes quorum sensing (QS) to regulate genetic transformation, virulence, and biofilm formation. The competence-stimulating peptide (CSP) is a 17-amino acid signal peptide that is used by S. pneumoniae to trigger QS. S. pneumoniae strains can be divided into two main specificity groups based on the CSP signal they produce (CSP1 or CSP2) and their compatible receptors (ComD1 or ComD2, respectively). Modulation of QS in S. pneumoniae can be achieved by targeting the CSP:ComD interaction using synthetic CSP analogues. However, to rationally design CSP-based QS modulators with enhanced activities, an in-depth understanding of the structural features that are required for receptor binding is needed. Herein, we report a comprehensive in-solution three-dimensional structural characterization of eight CSP1 and CSP2 analogues with varied biological activities using nuclear magnetic resonance spectroscopy. Analysis of these structures revealed two distinct hydrophobic patches required for effective ComD1 and ComD2 binding.

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection.

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.

Structure of RNA Stem Loop B from the Picornavirus Replication Platform.

The presumptive RNA cloverleaf at the start of the 5'-untranslated region of the picornavirus genome is an essential element in replication. Stem loop B (SLB) of the cloverleaf is a recognition site for the host polyC-binding protein, which initiates a switch from translation to replication. Here we present the solution structure of human rhinovirus isotype 14 SLB using nuclear magnetic resonance spectroscopy. SLB adopts a predominantly A-form helical structure. The stem contains five Watson-Crick base pairs and one wobble base pair and is capped by an eight-nucleotide loop. The wobble base pair introduces perturbations into the helical parameters but does not appear to introduce flexibility. However, the helix major groove appears to be accessible. Flexibility is seen throughout the loop and in the terminal nucleotides. The pyrimidine-rich region of the loop, the apparent recognition site for the polyC-binding protein, is the most disordered region of the structure.

Mechanism of Histone H3K4me3 Recognition by the Plant Homeodomain of Inhibitor of Growth 3.

Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (KD ranging between 0.63 and 0.93 µm) and is about 200-fold stronger than with the unmodified histone H3. NMR and computational studies revealed an aromatic cage composed of Tyr-362, Ser-369, and Trp-385 that accommodate the tri-methylated side chain of H3K4. Mutational analysis confirmed the critical importance of Tyr-362 and Trp-385 in mediating the ING3PHD-H3K4me3 interaction. Finally, the biological relevance of ING3PHD-H3K4me3 binding was demonstrated by the failure of ING3PHD mutant proteins to enhance ING3-mediated DNA damage-dependent cell death. Together, our results reveal the molecular mechanism of H3K4me3 selection by the ING3PHD and suggest that this interaction is important for mediating ING3 tumor suppressive activities.

Structure and Function of the PriC DNA Replication Restart Protein.

Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks.

Simplified AIP-II Peptidomimetics Are Potent Inhibitors of Staphylococcus aureus AgrC Quorum Sensing Receptors.

The bacterial pathogen Staphylococcus aureus controls many aspects of virulence by using the accessory gene regulator (agr) quorum sensing (QS) system. The agr system is activated by a macrocyclic peptide signal known as an autoinducing peptide (AIP). We sought to develop structurally simplified mimetics of AIPs for use as chemical tools to study QS in S. aureus. Herein, we report new peptidomimetic AgrC receptor inhibitors based on a tail-truncated AIP-II peptide that have almost analogous inhibitory activities to the parent peptide. Structural comparison of one of these peptidomimetics to the parent peptide and a highly potent, all-peptide-derived, S. aureus agr inhibitor (AIP-III D4A) revealed a conserved hydrophobic motif and overall amphipathic nature. Our results suggest that the AIP scaffold is amenable to structural mimicry and minimization for the development of synthetic agr inhibitors.

The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data.

We introduce AUDANA (Automated Database-Assisted NOE Assignment), an algorithm for determining three-dimensional structures of proteins from NMR data that automates the assignment of 3D-NOE spectra, generates distance constraints, and conducts iterative high temperature molecular dynamics and simulated annealing. The protein sequence, chemical shift assignments, and NOE spectra are the only required inputs. Distance constraints generated automatically from ambiguously assigned NOE peaks are validated during the structure calculation against information from an enlarged version of the freely available PACSY database that incorporates information on protein structures deposited in the Protein Data Bank (PDB). This approach yields robust sets of distance constraints and 3D structures. We evaluated the performance of AUDANA with input data for 14 proteins ranging in size from 6 to 25 kDa that had 27-98 % sequence identity to proteins in the database. In all cases, the automatically calculated 3D structures passed stringent validation tests. Structures were determined with and without database support. In 9/14 cases, database support improved the agreement with manually determined structures in the PDB and in 11/14 cases, database support lowered the r.m.s.d. of the family of 20 structural models.

Probabilistic validation of protein NMR chemical shift assignments.

Data validation plays an important role in ensuring the reliability and reproducibility of studies. NMR investigations of the functional properties, dynamics, chemical kinetics, and structures of proteins depend critically on the correctness of chemical shift assignments. We present a novel probabilistic method named ARECA for validating chemical shift assignments that relies on the nuclear Overhauser effect data . ARECA has been evaluated through its application to 26 case studies and has been shown to be complementary to, and usually more reliable than, approaches based on chemical shift databases. ARECA is available online at http://areca.nmrfam.wisc.edu/.

Integrative NMR for biomolecular research.

NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps associated with its practice have made the approach daunting for non-specialists. We introduce an NMR platform that makes biomolecular NMR spectroscopy much more accessible by integrating tools, databases, web services, and video tutorials that can be launched by simple installation of NMRFAM software packages or using a cross-platform virtual machine that can be run on any standard laptop or desktop computer. The software package can be downloaded freely from the NMRFAM software download page ( http://pine.nmrfam.wisc.edu/download_packages.html ), and detailed instructions are available from the Integrative NMR Video Tutorial page ( http://pine.nmrfam.wisc.edu/integrative.html ).

Characterization of structural elements in native autoinducing peptides and non-native analogues that permit the differential modulation of AgrC-type quorum sensing receptors in Staphylococcus aureus.

Staphylococcus aureus uses short macrocyclic peptides (i.e., autoinducing peptides, or AIPs) to assess its local population density in a cell-cell signaling mechanism called quorum sensing (QS). At high cell numbers, this pathogen can initiate many virulent behaviors that allow for the establishment of infection. Binding of the AIP signal to its cognate transmembrane AgrC-type receptor is a critical event in the QS signaling cascade; consequently, interference of AIP:receptor interactions may have the potential to prevent and eradicate certain S. aureus infections. To date, four pairs of AIP:AgrC receptors have been identified in S. aureus, each pair being utilized by a specific S. aureus group (I-IV). Other staphylococcal species also use closely related, but distinct, AIP:AgrC pairs to control QS. We seek to develop non-native ligands capable of intercepting AIP:AgrC binding in each S. aureus group and in related species. As these bacteria may use their respective AIP signal to attenuate the QS systems of other groups/species, such ligands would provide valuable chemical tools to probe possible interference mechanisms in a range of contexts. In the current study, we used solution-phase NMR techniques to characterize the 3-D structures of a set of known native and non-native peptides that have differential modulatory activity in certain AgrC receptors. Analysis of these structures revealed several distinct structural motifs that belay differential activity in selected S. aureus AgrC receptors (i.e., AgrC-I, AgrC-II, and AgrC-III). The results of this study can be leveraged for the design of new synthetic ligands with enhanced selectivities and potencies for these AgrC receptors.

Structural basis for the photoconversion of a phytochrome to the activated Pfr form.

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red-light-absorbing, ground state (Pr) and a far-red-light-absorbing, photoactivated state (Pfr). Although the structures of several phytochromes as Pr have been determined, little is known about the structure of Pfr and how it initiates signalling. Here we describe the three-dimensional solution structure of the bilin-binding domain as Pfr, using the cyanobacterial phytochrome from Synechococcus OSB'. Contrary to predictions, light-induced rotation of the A pyrrole ring but not the D ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intradomain and interdomain contact sites within the phytochrome dimer. On the basis of our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.

Highly Stable, Amide-Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus.

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP-III) signal that can inhibit AgrC-type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide-linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.

Dynamic structural changes underpin photoconversion of a blue/green cyanobacteriochrome between its dark and photoactivated states.

The phytochrome superfamily of photoreceptors exploits reversible light-driven changes in the bilin chromophore to initiate a variety of signaling cascades. The nature of these alterations and how they impact the protein moiety remain poorly resolved and might include several species-specific routes. Here, we provide a detailed picture of photoconversion for the photosensing cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) domain from Thermosynechococcus elongatus (Te) PixJ, a member of the cyanobacteriochrome clade. Solution NMR structures of the blue light-absorbing dark state Pb and green light-absorbing photoactivated state Pg, combined with paired crystallographic models, revealed that the bilin and GAF domain dynamically transition via breakage of the C10/Cys-494 thioether bond, opposite rotations of the A and D pyrrole rings, sliding of the bilin in the GAF pocket, and the appearance of an extended region of disorder that includes Cys-494. Changes in GAF domain backbone dynamics were also observed that are likely important for inter-domain signal propagation. Taken together, photoconversion of T. elongatus PixJ from Pb to Pg involves complex structural changes within the GAF domain pocket that transduce light into a mechanical signal, many aspects of which should be relevant to others within the extended phytochrome superfamily.

NMRFAM-SDF: a protein structure determination framework.

The computationally demanding nature of automated NMR structure determination necessitates a delicate balancing of factors that include the time complexity of data collection, the computational complexity of chemical shift assignments, and selection of proper optimization steps. During the past two decades the computational and algorithmic aspects of several discrete steps of the process have been addressed. Although no single comprehensive solution has emerged, the incorporation of a validation protocol has gained recognition as a necessary step for a robust automated approach. The need for validation becomes even more pronounced in cases of proteins with higher structural complexity, where potentially larger errors generated at each step can propagate and accumulate in the process of structure calculation, thereby significantly degrading the efficacy of any software framework. This paper introduces a complete framework for protein structure determination with NMR--from data acquisition to the structure determination. The aim is twofold: to simplify the structure determination process for non-NMR experts whenever feasible, while maintaining flexibility by providing a set of modules that validate each step, and to enable the assessment of error propagations. This framework, called NMRFAM-SDF (NMRFAM-Structure Determination Framework), and its various components are available for download from the NMRFAM website (http://nmrfam.wisc.edu/software.htm).