"Solution NMR studies provide structural basis for endotoxin pattern recognition by theinnate immune receptor CD14"

Seth Albright, Bin Chen, Kristen Holbrook, Nitin U. Jain
The University of Tennessee, Knoxville

Sepsis and septic shock are clinical manifestations of an overactivated innate immune response to outer cell wall components known as endotoxins from gram-negative and gram-positive bacteria. Endotoxin recognition by the innate immune receptor CD14 during initiation of sepsis has been investigated for a long time, however, the structural basis for differential endotoxin pattern recognition by CD14 is unclear due to non-availability of high resolution structural data for the CD14-endotoxin complexes. Here, we present results from a direct structural observation of the binding interaction between CD14 and two endotoxin ligands, Lipopolysaccharide (LPS) and Lipoteichoic acid (LTA), from gram-negative and gram-positive bacteria, which may help explain the broad specificity of CD14 in endotoxin pattern recognition. Using multidimensional solution NMR spectroscopy, a high-resolution NMR structure for the deep rough mutant of LPS from E. coli, ReLPS, has been determined for the first time, and structural analysis of its complex with CD14 reveals the specific LPS molecular patterns recognized by CD14. Similar structural studies have been carried out for LTA from B. subtilis to identify its CD14 binding motifs. Comparison of structural data for ReLPS and LTA indicates that endotoxin binding is primarily driven by specific contacts that the endotoxin acyl chains make with the leucine-rich repeats in the hydrophobic pocket of CD14, while specificity of endotoxin recognition is likely modulated by interaction of the lipid A sugar moeties with charged residues in the hydrophilic rim of CD14. This is also confirmed by NMR studies of isotopically labeled CD14 with the same endotoxins, which constitutes the first detailed structural characterization of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding.

"De novo high-resolution protein structure determination from sparse spin-labeling EPR data"

Nathan Alexander
Vanderbilt University

Nuclear magnetic resonance (NMR) and X-ray crystallography have resulted in over 99.99% of all protein structures currently deposited in the protein data base, but membrane proteins remain difficult targets for both techniques. Hence, membrane protein structure determination remains one key challenge in structural biology which must be addressed with alternative techniques. Accordingly, electron paramagnetic resonance (EPR) can be used to investigate membrane proteins that are not amenable to NMR and X-ray crystallography by measuring residue exposure to membrane or solvent and determining intramolecular distances of up to 50 Å. However, EPR data alone cannot produce a high-resolution protein structure. By coupling EPR datasets with computational protein structure prediction, atomic detail protein structure determination of previously inaccessible proteins becomes feasible.

"Structure-Based Development of Novel Dihydropteroate Synthase Inhibitors "

Katherine Ayers
St. Jude Children's Research Hospital

The bacterial enzyme dihydropteroate synthase, or DHPS, catalyzes the addition of p-aminobenzoic acid (pABA) to dihydropterin pyrophosphate (DHPP) to form pteroic acid as a key step in bacterial folate biosynthesis. It is targeted by the sulfonamide class of antibiotics, and most sulfonamide resistance is due to mutations of the DHPS gene. Sulfonamides bind in the pABA pocket of the enzyme, and resistance mutations cluster in two flexible loops that presumably engage this pocket during catalysis. Using structure-based drug design approaches, virtual screening and biochemical assays, we are searching for novel inhibitors of this validated antibacterial target that engage the second pocket at the active site that binds the pterin substrate. This second pocket is vital to the structural integrity of the protein and therefore less able to accommodate resistance mutations. An important goal of our inhibitor design studies is to understand the catalytic mechanism, and this understanding will also provide insights into the structural basis of sulfonamide resistance. Here we report our recent results on this broadly based drug discovery program that encompasses (1) biochemistry and enzyme kinetics, (2) protein crystallography, (3) virtual screening and SAR analyses, and (4) catalytic mechanism.

"RPA Quaternary Remodeling: Insights into a DNA-Processing Machine"

Chris Brosey
Vanderbilt University

Replication Protein A (RPA) serves as the primary ssDNA-binding protein in eukaryotes, playing a pivotal role in the protection and organization of ssDNA during DNA replication, damage response and repair. RPA's ability to participate in such a diversity of events is thought to arise in part from its modular domain structure, though little is currently known about its architecture or how its quaternary organization might facilitate its role in DNA processing. NMR spectroscopy on intact 116 kDa RPA and a number of multi-domain constructs provides evidence of a highly mobile and dynamic view of RPA quaternary structure, consistent with RPA's ability to coordinate actions within multi-protein DNA processing machinery.

"Effect of temperature and concentration of glycerol-water binary mixtures on the dynamics of proteins"

Pavan Ghatty
Oak Ridge National Laboratory

Glycerol has been implicated as a key player in the freeze-tolerance exhibited by some species of frogs. As winter advances, the concentration of glycerol in these frogs increases. The decrease in temperature leads to freezing of their bodily parts from the skin to the heart which eventually stops functioning at the peak of winter. Although the effect of temperature on protein-water and protein-glycerol mixtures has been well studied using various experimental techniques and simulations, the interplay between changing temperature and concentration of glycerol-water mixtures on the dynamics of proteins has not been well studied. We have employed molecular dynamics simulations to study this effect. We observe that the hydrogen bonded network between the various components is a good indicator of the dynamics. The relaxation time of these networks increases with a decrease in temperature and/or an increase in the concentration of glycerol. These effects can be explained by the number of hydrogen bonds that an average water molecule makes. One can thus control the dynamics of a protein by simply varying the behavior of solvent.

"How PUMA Attacks Its Prey: Understanding the Molecular Basis of PUMA-Induced Cell Death "

R. W. Kriwacki
St. Jude Children's Research Hospital

PUMA, a member of the pro-apoptotic BH3-only proteins, has been shown to be essential for p53 induced apoptosis in response to genotoxic stress, but the mechanism(s) through which PUMA induces mitochondrial outer membrane permeabilization (MOMP) has been subject to extensive study and much debate. Here we show that PUMA is an intrinsically unstructured protein free in solution. While PUMA is completely devoid of tertiary structure, we show using NMR and circular dichroism spectroscopy that the 25 residue segment corresponding to the BH3 domain exhibits slightly helical propensity in solution. Furthermore, we report the 2.9 Å crystal structure of a 25 amino acid PUMA BH3 domain peptide bound to the anti-apoptotic BCL-xL protein. The PUMA BH3 domain undergoes a disorder-to-order transition upon binding BCL-xL and assumes a rigid helical conformation. Reminiscent of previously determined BCL-xL/BH3 structures, the structure of the BCL-xL surface-exposed hydrophobic groove changes to accommodate the PUMA BH3 helix; however, the extent of structural rearrangement seen for BCL-xL in the PUMA bound crystal structure differs quite drastically from the other BCL-xl/BH3 structures. The results of our structural studies of PUMA and the PUMA/BCL-xL complex provide important insights into the molecular basis of p-53-dependent PUMA-induced apoptosis and into the mechanistic basis regarding the BCL-xL, p53 PUMA tripartite nexus.

"The C-terminus of Dishevelled folds back to its own PDZ domain: Implication of inactive state of Dishevelled"

Ho-Jin Lee
St. Jude Children's Research Hospital

The Wnt signaling pathways play crucial roles in stem cells, embryo development, and maintenance of adult cells. The signaling has been thought to be regulated by Dishevelled (Dvl/Dsh) protein. The regulatory mechanism of how Dvl activates the signaling remains unknown. Using NMR spectroscopy and biophysical methods, we report here that the extreme C-terminus of Dvl associates with its own PDZ domain, indicating that Dvl has auto-inhibited conformation. In Xenopus, the C-terminally truncated Xdsh (XdshΔC, no PDZ-binding motif), but not wild-type Xdsh, gain of function leads to disruption of convergent extension movement in Xenopus embryos and in activin-treated ectodermal explants. The results suggest that the auto-inhibited conformation of Dvl would play a crucial role in the modulation of the Wnt signaling pathways.

"Cryo-EM guided de novo Protein Fold Elucidation"

Steffen Lindert
Vanderbilt University

Cryo-electron microscopy (cryo-EM) is an increasingly important technique to elucidate the structure of large macromolecular assemblies such as whole viruses. Cryo-EM generally does not provide atomic resolution information, but numerous sub-nanometer resolution structures have been reported in the last years. At 7 Å resolution helices are observed as density rods but strand and coil regions are not discernable. The goal of this project is the development of a computational protein structure prediction algorithm that incorporates the experimental cryo-EM data as restraints. The placement of helices is restricted to regions where density rods are observed in the cryo-EM density map. The Monte Carlo based search algorithm is driven by knowledge based energy functions.

This method has been benchmarked with ten highly α-helical proteins of known structure. The chosen proteins range in size from 250 to 350 residues. Starting with knowledge of the true secondary structure for these ten proteins, the method can identify the correct topology within the top scoring 10 models. With more realistic secondary structure prediction information, the correct topology is found within the top scoring 150 models for seven of the ten proteins. These results prove the effectiveness of the proposed assembly algorithm. In a next step the algorithm will be applied to one of the proteins in human adenovirus, protein IIIa. This protein, for which there is no high resolution structure, is predicted to be highly α-helical and is resolved in a 6.9 Å resolution cryoEM adenovirus structure as a bundle of ~13 α-helical density rods.

"Virtual High Throughput Screening for mGluR5 potentiators"

Ralf Mueller
Vanderbilt University

Metabotropic glutamate receptors play a key role in modulating synaptic activity in the Central Nervous System. As such they represent viable targets for treating diseases like schizophrenia, ParkinsonÕs disease, and others. mGluR5 potentiators modulate the response of mGluR5 to agonists. Starting from a set of assay data for mGluR5 potentiators, an artificial neural network was trained to predict the mGluR5 potentiation (EC50) for small molecules. The input data consists of scalar molecular descriptors, autocorrelation functions, and radial distribution functions. The enrichment factor on the original screening data was calculated to be 43 (from approximately 1% compounds being active to 43%). After refining the artificial neural network, it was applied to scan a database of 450,000 commercially available drug candidates (ChemBridge) for new possible leads. A set of 800 compounds was selected for their high predicted activity to be tested at the High Throughput Screening center at Vanderbilt. The talk presents the methods developed and implemented to train this artificial neural network and discusses the results of this test.

"Hydrogen-Deuterium Exchange Studies of a 30 kDa Antibiotic Resistance Conferring Enzyme"

Adrianne L. Norris
The University of Tennessee, Knoxville

In recent years, the medical world has seen an alarming increase in antibiotic resistance among numerous classes of bacteria. While different mechanisms exist to hinder the effectiveness of various antibiotics, one of the most common is enzyme assisted covalent modification of aminoglycosides. Aminoglycosides are broad spectrum antibiotics that target 16S RNA of the 30S ribosomal subunit found only in bacteria. This inhibits protein translation leading to cell death. The 30 kDa enzyme aminoglycoside-phosphotransferase(3')-IIIa (APH) from Enterococcus faecalis, a drug resistant strain that is a common cause of severe hospital infections, is capable of binding to a broad range of aminoglycosides and causing a detoxifying phosphorylation event at the 3'- or 5"-OH groups via a metal-ATP complex. Hydrogen-deuterium exchange studies by nuclear magnetic resonance (NMR) have shown that APH is very flexible in solution when in the apo state and may be even intrinsically unstructured. A well-defined structure is acquired upon aminoglycoside binding and a number of amino acids distant from the active site are more protected from the solvent when neomycin B is the substrate relative to kanamycin A suggesting a global structural adjustment for broad antibiotic selectivity. Furthermore, it was discovered that amino acids in and around the nucleotide binding site become less protected from solvent in the presence of magnesium and the ATP analog, AMPPCP. Several of these residues are situated in a five stranded anti-parallel beta sheet suggesting that the sheet stabilizing hydrogen bonds involving their amides are weakened or broken as a result of nucleotide binding. This may be attributed to the stacking interaction between the nucleotideÕs adenine ring and the side chain of tyrosine 42. Detailed structural analysis of APH-antibiotic interactions provides critical information about the dynamics of this enzyme and may give insight into other aminoglycoside modifying proteins. This would create a more intelligent foundation in designing drugs to combat the antibiotic resistance problem.

"Allosteric interplay in Thyroid hormone receptor transactivation"

Balananda Durjati Putcha
The University of Tennessee, Knoxville

Thyroid hormone receptor (TR) is a ligand mediated transcription factor that plays an important role in growth, development and homeostasis in ammals. Transactivation mediated by the thyroid receptor is an integrated response to diverse allosteric stimuli including ligands, the heterodimeric partner, Retinoid X receptor (RXR), co-regulators and the DNA response elements (TREs). In order to dissect the regulatory mechanism of TR activity by various allosteric stimuli, we used in vitro reconstituted core transactivation complex. Allosteric effect of each component of the complex on the ability to recruit a coactivator peptide, SRC1 or TREs by TR was quantified systematically using Isothermal titration calorimetry (ITC). These changes in the binding properties are related to distinct quantitative activation properties mediated by RXR:TR heterodimers in vivo. SRC1 recruitment profile in response to T3 and/or 9c is similar to activity profile with those ligands in vivo. Therefore ligand induced RXR:TR transactivation is regulated in part by levels of coactivator recruitment, through inter-receptor crosstalk. Binding of TREs at DNA binding domain (DBD) exerts profound effect on the SRC1 recruitment by TR at Ligand binding domain (LBD), depending upon the presence of RXR. Similarly, subject to the presence of RXR, SRC1 binding at LBD enhances recruitment of TREs by DBD through intra-receptor allosteric regulation. Finally, modulation of quantitative response on different TREs observed in vivo may be a consequence of differences in the thermodynamic properties of TREÐRXR:TR interactions. Our approach using in vitro reconstituted nuclear receptor transactivation complex gives mechanistic insights into the allosteric interplay, in the absence of structural information for such multi-component systems.

"A New Protein Architecture for Processing Alkylation Damaged DNA: The Crystal Structure of DNA Glycosylase AlkD"

Emily Rubinson
Vanderbilt University

DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. DNA glycosylases active toward alkylation-damaged bases include enzymes that are highly specific for 3-methyladenine and others that can recognize a variety of alkylpurines. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases.

"Computational Studies of Drug Resistance of H5N1 Influenza Viruses to Oseltamivir"

Xuelin Wang
St. Jude Children's Research Hospital

The H5N1 influenza A virus is one of the largest pandemic threats in the world today. Two well-known neuraminidase (NA) inhibitors, oseltamivir and zanamivir effectively prevent the spread of influenza infection. However, the emergence of drug-resistant viruses diminishes the drugsÕ effectiveness. Drug resistance depends strongly on the binding properties of the virus-drug complexes. Key residue mutations within the active site of the viral NA glycoproteins can diminish binding, resulting in drug resistance. We performed computational molecular modeling and simulation methods to characterize the molecular mechanism of H5N1 resistance to oseltamivir and to predict future potential drug-resistant H5N1 mutations. We examined two oseltamivir-resistant H5N1 mutations, H274Y and N294S, and one H3N2 mutation, E119G, as a control. Six-nanosecond unrestrained molecular dynamic simulations with explicit solvent were performed using virus-oseltamivir complexes containing either wild-type H5N1 or a variant. MM/PBSA techniques were then used to rank the binding free energies of these complexes. Detailed structure and energy analyses indicated that changes in the pocket 1 region of NA are a key source of drug resistance of H5N1 mutants.

"p27^Kip1 Phosphorylation, Degradation and Cancer"

Yuefeng Wang
St. Jude Children's Research Hospital

p27^Kip1 blocks cell cycle progression at the G₁-S transition by potently inhibiting cyclin-dependent kinases (Cdks) that are the master timekeepers of cell division. Tyrosine phosphorylation by non-receptor tyrosine kinases (NRTKs) regulates p27 ubiquitination and proteasomal degradation at the G1-S boundary and this mechanism has been shown to be upregulated in several types of cancer. For example, we recently demonstrated that phosphorylation of tyrosine 88 (Y88) by Abl kinase promotes degradation of p27 in chronic myelogenous leukemia (CML) cells (Cell, 2007, 128, 269-280). Here we will present the biochemical and structural mechanism by which phosphorylation of two tyrosine residues, Y74 and Y88, by Src kinase re-activates cyclin-dependent kinase 2 (Cdk2) and promotes degradation of p27 in breast cancer cells. Previous studies showed that phosphorylation of p27 on Y88 partially reactivated Cdk2 within otherwise inhibited p27/Cdk2/cyclin A complexes. New results indicate that phosphorylation of both Y74 and Y88 of p27 by Src reactivates Cdk2 (within p27/Cdk2/cyclin A complexes) to a greater extent than does phosphorylation of Y88 alone. In contrast to phosphorylation of Y88 alone, NMR studies suggest that phosphorylation of Y74 and Y88 by Src dislodges a large segment of p27 from its normal binding site on the N-terminal lobe of Cdk2. We propose that this not only ejects inhibitory Y88 from the Cdk2 active site but also allows the N-terminal lobe of Cdk2 to assume a more native-like conformation, accounting for the largely restored catalytic activity. Dual tyrosine phosphorylation (on Y74/Y88) enhances phosphorylation of threonine 187 (T187) of p27 by Cdk2/cyclin A, which promotes SCF-Skp2-dependent ubiquitination and degradation of p27. Preliminary results suggest that phosphorylation of tyrosine residues of p27 may occur sequentially, with phosphorylation of Y88 promoting that of Y74. This study illustrates how p27 serves to integrate proliferative signals from NRTKs and strengthens the link between p27 loss due to tyrosine phosphorylation and cancer in humans.

"Knowledge based energy functions assist in ab initio protein structure prediction"

Nils Woetzel
Vanderbilt University

Elucidating structures of proteins is the central task in understanding biological pathways. X-ray crystallography and nuclear magnetic resonance experiments significantly contribute to the effort of determining protein structures, but are applicable to all proteins. Advances in computational protein structure prediction and improvement in today's computational resources makes ab initio structure prediction feasible. A energy potentials, derived from known protein structures will be presented, that can filter for native like protein structures in a set of computationally generated protein models for a given amino acid sequence.