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NMR Protein Solution Structures

B. subtilis Acyl Carrier Protein (ACP)

Acyl carrier protein (ACP) is a fundamental component of fatty acid biosynthesis where the fatty acid chain is elongated by the fatty acid synthetase system while attached to the 4'-phosphopantetheine prosthetic group (4'-PP) of ACP. Activation of ACP is mediated by holo-acyl carrier protein synthase (ACPS) where ACPS transfers the 4'-PP moiety from coenzyme A (CoA) to Ser-36 of apo-ACP. Both ACP and ACPS have been identified as essential for E. coli viability and potential targets for development of antibiotics.

Structural data was collected for both the apo- and holo-forms of ACP that suggest that the two forms of ACP are essentially identical. Comparison of the published structures for E. coli ACP and Actinorhodin Polyketide Synthase acyl carrier protein (act apo-ACP) from Streptomyces coelicolor A3(2) with B. subtilis ACP indicates similar secondary structure elements, but an extremely large rms deviation between the three ACP structures (> 4.3 angstrom). The structural difference between B. subtilis ACP and both E. coli and act apo-ACP is not attributed to an inherent difference in the proteins but is probably a result of a limitation in the methodology available for the analysis for E. coli and act apo-ACP. Comparison of the structure of free ACP with the bound form of ACP in the ACP-ACPS complex reveals a displacement of helix II in the vicinity of Ser-36. The induced perturbation of ACP by ACPS positions Ser-36 proximal to coenzyme A and aligns the dipole of helix II to initiate transfer of 4'-PP to ACP.

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A. fulgidis Peptidyl-tRNA Hydrolase (NESG AF2095, GR4)

The thermophilic archaea Archaeglobus fulgidis AF2095 protein is an example of a protein of unknown biological function targeted for structural analysis by the Northeast Structural Genomics Consortium (NESG), which also belongs to the previously unannotated Pfam family UPF0099. Comparison of the 3D NMR structure of A. fulgidis AF2095 and the 1.95 angstrom X-ray crystal structure of the functionally-unannotated thermophilic archaea T. acidophilum protein TA0108 that has recently been deposited in the Protein Database (PDB: 1rlk) with the human Pth2 X-ray structure suggest that these proteins are also Pth2 enzymes.

Peptidyl-tRNA hydrolase (Pth) is a crucial bacterial enzyme. Peptidyl-tRNA hydrolase cleaves the peptide from peptidyl-tRNA molecules that have prematurely dissociated from the ribosome during protein translation. The freed tRNA can then be recycled back into the protein synthesis process. Aminoacyl tRNA-fMet, the initiator of the translation process, is a relatively poor substrate for E. coli Pth and is correspondingly protected from hydrolysis. This permits free aminoacyl tRNA-fMet to readily participate in the formation of the relatively slow ribosomal initiation complex without being prematurely hydrolyzed.

Although it had been proposed that the Pth2 family has no bacterial members, assessments of homology models based on the 3D structure of AF2095 reported here demonstrates a wide phylogenetic distribution of the Pth2 enzyme class, including many bacterial proteins, consistent with the recent inclusion of bacterial members in Pfam family UPF0099. Phylogenic analysis of the Pth2 enzymes homologous to AF2095 supports convergent evolution of the Pth and Pth2 enzymes suggesting that eukaryotes inherited Pth2 as part of the mitochondria organelle.

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S. aureus C-terminal domain of primase (DnaG)

The protein-protein interaction between the C-terminal domain (CTD) of primase (DnaG) and N-terminal domain of helicase (DnaB) is essential for DNA synthesis during bacterial DNA replication. This interaction is conserved in all bacteria, and distinctly different from that of eukaryotes making it an attractive antibiotic target. To develop this interface as an antibiotic target and ligand-binding site, we determined the solution structure and dynamics analysis of DnaG CTD from Staphylococcus aureus. Its structure is more similar to the one from Geobacillus stearothermophilus than from Escherichia coli, consistent with a structural divergence between Firmicutes and Proteobacteria. The greatest divergence lies in the final two α-helices that form the C2 subdomain. NMR measurements indicate that the two subdomains are hold rigidly with respect to one another and that all relative movement is focused on a single residue N564 in S. aureus. The larger subdomain has regions undergoing rapid folding and unfolding but the C2 subdomain has a very high order. The model that emerges from this analysis is that 5 residues in the inflexible C2 subdomain make contact with one of a pair of DnaB N-terminal domains. Next two residues in the partially unfolded C1 subdomain make contact with the other DnaB NTD in a way that stimulates ATPase activity. Since C2 subdomain-NTD interaction is species-dependent, it is the preferred target area.

The primase CTD structure is composed of 8 helices arranged into two subdomains as is the primase CTD NMR structure from G. stearothermophilus. In our S. aureus primase CTD structure, the first six helices create the C1 subdomain and encompass residues 467 to 565. The last two helices, 7 and 8, create the C2 subdomain encompassing residues 572 to 603. When the 20 structures are overlaid using the alignment of the C1 subdomain backbone residues as a guide, the C2 subdomain structures do not superimpose. The converse overlay based on the alignment of the C2 subdomain shows the same effect. The poor superimposition arises because of the very low number of structural restraints between the subdomains. The structure dynamics indicates the subdomains behave as two independent domains. This is further supported by the observation that the loop region between the subdomains lack sequential NH-NH NOEs and the loop residues, G567, Q568 and E569, exhibit exchange peaks. These residues are likely conformationally dynamic and undergoing rapid exchange with the solvent, a feature indicative of exposed residues that lack protection from hydrogen bonds found in secondary structures.

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Human DNAJ Homologue Subfamily A Member 1 (DNAJA1, NESG HR30991)

The human protein DnaJ homolog subfamily A member 1 (DNAJA1) was previously shown to be down-regulated five-fold in pancreatic cancer cells, and has been targeted as a biomarker for pancreatic cancer. But, little is known about the specific biological function for DNAJA1 or the other members of the DnaJ family encoded in the human genome. Our results suggest the overexpression of DNAJA1 suppresses the stress response capabilities of the oncogenic transcription factor, c-Jun, and results in the diminution of cell survival. DNAJA1 likely activates a DnaK protein by forming a complex that suppresses the JNK pathway, the hyperphosphorylation of c-Jun, and the anti-apoptosis state found in pancreatic cancer cells. A high-quality NMR solution structure of the J-domain of DNAJA1 combined with a bioinformatics analysis and a ligand affinity screen identifies a potential DnaK binding site, which is also predicted to overlap with an inhibitory binding site suggesting DNAJA1 activity is highly regulated.

The secondary structure and fold for DNAJA1-JD are similar to other J-domains found in DnaJ homologs. In effect, our DNAJA1-JD structure adopts the characteristic J-domain found in most species. The structure consists of four α-helices: residues 17-21 (α1); 29-42 (α2); 52-65 (α3); and 68-75 (α4). The loop between &alpha2 and α3 (residues 43-51) contains the highly conserved His-Pro-Asp (HPD) motif (residues 44-46). The DNAJA1-JD NMR structure combined with a bioinformatics analysis and a ligand affinity screen identified a potential DnaK-like binding site and TIM16-like inhibitory binding site. Interestingly, both of these binding sites involve the conserved α2-helix and suggest DNAJA1 activity is highly regulated.

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Human Basic Fibroblast Growth Factor (FGF-2)

Basic fibroblast growth factor (FGF-2), a member of a protein family that includes three oncogenes (FGF-3, FGF-4 and FGF-5), exhibits angiogenic and a variety of growth and differentiation activities. Its diverse role in regulating cell growth and differentiation has suggested an involvement in wound healing, tumor growth and cancer. A common feature of the FGF family members is their high affinity toward heparin sulfate proteoglycans (HSPG). The interaction of FGF-2 with HSPG is required for high-affinity binding to its cell surface tyrosine kinase receptor (FGFR) and essential for mediating internalization and intracellular targeting through a proposed mechanism of receptor dimerization. It has been suggested that HSPG might interact directly with FGFR to facilitate the formation of a trimolecular complex and that the HSPG induced dimerization of FGF-2 may be important for receptor dimerization. Near complete 1H, 15N, 13CO, and 13C assignments, solution secondary structure, dynamics, high-resolution structure of FGF-2 and its interaction with heparin have been analyzed by NMR. A helix-like structure was observed for residues 131-136 which is part of the heparin binding site (residues 128-138). The discovery of the helix-like region in the primary heparin binding site instead of the β-strand conformation described in the x-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. A total of seven tightly bound water molecules were found in the FGF-2 structure, two of which are located in the heparin binding site. Presented the first direct experimental evidence obtained by NMR, independently confirmed by dynamic light scattering and biological relevance established in cell-based and cell-free assays to propose a specifically oriented heparin-FGF-2 complex. Since the FGF-2-tetrasaccharide trans-dimer is inactive in receptor binding and initiation of the biological response, we conclude that the minimum active structural unit of the FGF-2-heparin complex is the properly oriented cis-dimer component of the tetramer "sandwich" motif induced by the decasaccharide.

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Human Interleukin-4 (IL-4)

Interleukin-4 (IL-4) is one of a group of cytokines that play a central role in the control and regulation of the immune and inflamatory systems. Specific activities associated with IL-4 are the stimulation of activated B cell, T lymphocyte, thymocyte, mast cell proliferation and the induction of cytotoxic CD8+ T cells. The latter is responsible for the antitumor activity of IL-4. Renal tumor cells that secrete large doses of IL-4 can establish tumor-specific immunity toward a preexisting renal cancer. The very potent antitumor activity of IL-4 at the primary tumour site is also associated with the elicitation of a localized inflammatory infiltrate. Further, IL-4 is responsible generating and sustaining in vivo IgE and IgG1 in the T cell-dependent immune response by causing immunoglobuilin class switching of activated B cells to igE and IdG1, respectively (IL-4 overview). In order to provide a structural basis for understanding the mode of action of IL-4 and its interaction with its cell surface receptor, the NMR assignments, secondary structure and high resolution structure of IL-4 have been determined by multidimensional NMR. The structure of IL-4 is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhead connections. The linker elements between the helices are formed by either long loops, small helical turns or short strands. The overall topology is remarkably similar to that of growth hormone and granulocyte-macrophage colony stimulating factor, despite the absence of any sequence homology, and substantial differences in relative lengths of the helices, the length and nature of the various connecting elements, and the pattern of disulfide bridges. These three proteins, however, bind to cell surface receptors belonging to the same hematopoietic superfamily, which suggests that interleukin-4 may interact with its receptor in an analogous manner to that observed in the crystal structure of the growth hormone-extracellular receptor complex.

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Human Interleukin-13 (IL-13)

Interleukin-13 has been implicated as a key factor in asthma, allergy, atopy and inflammatory response establishing the protein as a valuable therapeutic target. IL-13 is produced by activated T cells and promotes B cell proliferation, induces B cells to produce IgE, down regulates the production of proinflamatory cytokines, increases expression of VCAM-1 on endothelial cells, enhances the expression of class II MHC antigens and various adhesion molecules on monocytes. IL-13 mediates these functions through an interaction with its receptor on hematopoietic and other cell types, but currently no functional receptors have been identified on T cells. The signaling human IL-13 receptor (IL-13R) is a heterodimer composed of the interleukin-4 receptor a chain (IL-4Ra) and the IL-13 binding chain. The association of IL-13 with its receptor induces the activation of STAT6 (signal transducer and activation of transcription 6) and Janus-family kinase (JAK1, JAK2, TYK2) through a binding interaction with the IL-4Ra chain.

IL-13 shares many functional properties with IL-4 as a result of the common IL-4Ra component in their receptors. IL-4 exhibits a high affinity to IL-4Ra chain (KD = 20-300 pM) where this complex recruits the common g chain (gc) of IL-4R to form the signaling complex. Similarly, IL-13 binds to the IL-13 binding chain (IL-13Ra1) with relatively high affinity (KD ~ 4 nM) in the absence of the IL-4Ra chain, where an increase in affinity to IL-4R occurs in the presence of IL-4Ra (KD = 50 pM). IL-13 does not bind IL-4Ra in the absence of the IL-13 binding chain. As a result, IL-4 exhibits binding to both IL-4R and IL-13R due to the existence of the IL-4Ra chain in both receptors, but IL-13 does not bind IL-4R because of the absence of the IL-13 binding chain.

IL-13 and IL-4 are both members of the short chain four-helix bundle cytokine family. Despite the relatively low 25% sequence homology between IL-13 and IL-4, a similarity in the overall topology between the two proteins is expected. A combination of mutational and kinetic analysis has identified a distinct site on the IL-4 structure associated with IL-4Ra binding and a second site associated with signaling through the gc chain. Recently, the X-ray structure of IL-4 complexed with the ectodomain of IL-4Ra has been determined, which further defines the IL-4 - IL-4Ra interface. The resulting IL-13 NMR structure and assignments are consistent with previous short chain left-handed four-helix bundles where a significant similarity in the folding topology between IL-13 and IL-4 was observed. IL-13 shares a significant overlap in biological function with IL-4, a result of the common a chain component (IL-4Ra) in their respective receptors. Based on the available structural and mutational data, an IL-13/IL4Ra model and a sequential mechanism for forming the signaling heterodimer is proposed for IL-13.

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Human Class-I Histocompatibility Antigen-peptide Complexes (mAb TP25.99)

Human Class I histocompatibility antigen-peptide complexes initiate the cascade that results in the rejection of foreign tissue transplants. T cell receptor molecules bind the a1 and a2 domains of the HLA heavy chain in the antigen-peptide complex while the CD8 co-receptor binds the a3 domain to initiate a response. The anti-HLA Class I monoclonal antibody (mAb) TP25.99 has an unusual specificity as it recognizes determinants expressed on b2-m associated and b2-m free HLA Class I heavy chains. In the companion paper using phage display peptide libraries (Desai et al., 1998) we reported the identification of a cyclic and a linear peptide reacting with mAb TP25.99. The nineteen residue linear synthetic peptide sequence (X19) contains a stretch homologous to residues 239-242, 245 and 246 of HLA Class I heavy chains. The twelve residue cyclic peptide (LX-8) contains a stretch homologous to residues 194-198 of HLA Class I heavy chains. Analysis by two-dimensional transfer NOE spectroscopy of the induced solution structure of the X19 and LX-8 peptides in the presence of mAb TP25.99 showed that in spite of the lack of sequence homology, the two peptides adopt a similar structural motif. This motif corresponds to a short helical segment followed by a tight turn, reminiscent of the determinant loop region (residues 194-198) on b2-m associated HLA Class I heavy chains. An atomic rms distribution between the backbone atoms of X19 (residues 4-11) and LX-8 (residues 10-3) is 1.06 angstroms. The structural similarity between the X19 and LX-8 peptides and the lack of sequence homology suggests that mAb TP25.99 predominantly recognizes a structural motif instead of a consensus sequence. These results are also consistent with the observation that the association of HLA Class I heavy chains with b2-m modifies drastically their conformation and causes a change in their antigenic profile.

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Human Fibroblast Collagenase (MMP-1)

Fibroblast collagenase (MMP-1) is a member of the matrix metalloproteinase (MMP) family which include the collagenases, stromelysins and gelatinases. These enzymes require zinc and calcium for activity and are modular with both propeptide and catalytic domains being common to the entire family. The design of inhibitors of various MMPs for use as therapeutic agents in the treatment of arthritis and cancer has been an exceptionally active area of research. The MMPs are involved in the degradation of the extracellular matrix that is associated with normal tissue remodeling and, as result, MMP expression and activity is highly controlled by either specific inhibitors (tissue inhibitor of metalloendoproteases - TIMP), by cleavage of the inactive proenzyme or by transcription induction or suppression. A number of biochemical stimuli including cytokines, hormones, oncogene products and tumor promoters effect the synthesis and activation of MMPs. The apparent loss in this regulation can result in the pathological destruction of connective tissue and an ensuing disease state. The MMP family consists of more than 25 enzymes, and it has been postulated that the toxicity demonstrated by many MMP inhibitors in clinical trials may result from non-specific inhibition. Thus, the current approach relies on structure-based design of inhibitors of specific MMPs, where selectivity against MMP-1 may be a desirable trait. The extensive structural data available for the MMPs has enabled the identification of an obvious approach for designing specificity by taking advantage of the sequence difference and distinct size and shape of the S1' pocket. A number of examples have been previously reported using this approach. Nevertheless, the observed mobility of the MMP active site may complicate the design of potentially selective inhibitors.

We determined the first structure of a free MMP (MMP-1), where the dynamic analysis was first to indicate that the active site of MMPs are dynamic and flexible where a slow conformational exchange for residues comprising the active site (helix B, zinc ligated histidines and the nearby loop region) and a high mobility for residues P138-G144 in the vicinity of the active site for inhibitor-free MMP-1 was observed. Furthermore, analysis of inhibitors predicted to be selected against MMP-1 were surprisingly identified to bind tightly (picomolar) to the enzyme due to the observed dynamics by NMR of both the MMPs and the ligand. Numerous inhibitors complexed to both MMP-1 and MMP-13 have been determined utilizing a hybrid approach to quickly determine the corresponding co-structures. This was first described for the MMP-1:CGS-27023A structure.

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Human Collagenase 3 (MMP-13)

Human collagenase-3 (MMP-13) is a member of the matrix metalloproteinase (MMP) family which include the collagenases, stromelysins and gelatinases. The MMPs are involved in the degradation of the extracellular matrix which is associated with normal tissue remodeling processes such as pregnancy, wound healing, and angiogenesis. The MMPs have also demonstrated activity against cell surface and other pericellular non-matrix proteins further contributing to their cellular function. The MMP family consists of more than 25 enzymes where major discriminating factors are substrate preference (collagens, fibronectin, elastin, gelatins, etc.), domain structure and sequence alignment. The MMPs are modular proteins where a signal peptide, propeptide and catalytic domain are common to the entire family. Additional domains observed in MMP structures include fibronectin type II-like, hemopexin-like, vitronectin-like and transmembrane domains. Fundamental to the structural integrity and activity of MMPs is the presence of both zinc and calcium in the protein's structure. The active site zinc performs a critical function for both substrate binding and cleavage. Correspondingly, the design of MMP inhibitors has generally targeted the catalytic domain and active site zinc. The isolated catalytic domain maintains its general endopeptidase function but does not exhibit activity against its natural substrate. This is attributed to the absence of the hemopexin-like domain which is involved in substrate recognition and binding. The MMPs are a highly active set of targets for the design of therapeutic agents for the disease areas of arthritis and oncology. The MMPs have also been associated with multiple sclerosis, periodontitis, stroke, inflammatory bowel disease and cardiovascular disease. MMP expression and activity is highly controlled because of the degradative nature of these enzymes. The apparent loss in this regulation results in the pathological destruction of connective tissue and the ensuing disease state. MMP-13 was recently identified on the basis of differential expression in normal breast tissues and in breast carcinoma. In addition its expression has been reported in squamous cell carcinomas of the larynx, head and neck and HCS-2/8 human chondrosarcoma cells and during fetal ossification and in articular cartilage of arthritic patients. There have been a number of X-ray and NMR structures solved for the catalytic domain of MMPs complexed with a variety of inhibitors. There is a close similarity in the overall three-dimensional fold for these proteins consistent with the relatively high sequence homology (> 40%). Despite this similarity in the MMP structures there is distinct substrate specificity between these enzymes indicative of specific biological roles for the various MMPs and a corresponding association with unique disease processes. One example of this potential specificity is the over-expression of MMP-13 in breast carcinoma and MMP-1 in papillary carcinomas. Therefore the current paradigm in the development of MMP inhibitors is to design specificity into the structures of the small molecule instead of developing a broad spectrum MMP inhibitor. The rational behind this approach is that an inhibitor specific for the MMP uniquely associated with a disease process may potentially minimize toxic side effects. Comparison of the various MMP structures has identified a significant difference in the size and shape of the S1' pocket. This structural difference across the MMP family provides an obvious approach for designing specificity into potent MMP inhibitors by designing compounds that appropriately fill the available space in the S1' pocket while taking advantage of sequence differences.

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Human Oncostatin M NMR-based Homology Model (OM)

Oncostatin M (OM) is a member of cytokine family which regulates the proliferation and differentiation of a variety of cell types and includes interleukin-4 (IL-4), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and granulocyte-colony stimulating factor (G-CSF). This family of proteins adopt a four helix bundle fold with up-up-down-down topology and contain intramolecular disulfide bonds. The shared functions of these proteins are mediated through the interaction of the signal transducing membrane glycoprotein, gp130, with the extracellular domains of these cytokine receptors. Additional biological activities attributed to OM include the inhibition of several tumor cell lines, inhibition of embryonic stem cell differentiation a regulator of endothelial cells and the growth stimulation of several fibroblast cell lines. OM has also been shown to be a mitogen for AIDS-related Kaposi's sarcoma cells, where it functions as an autocrine growth factor.

Since an X-ray or NMR structure for OM is not currently available, a homology model for OM was determined from the X-ray structures of hGH, LIF and G-CSF where the alignment was based on the NMR secondary structure instead of sequence. The OM secondary structure was determined from NMR structural data and the secondary structure for hGH, LIF and G-CSF were obtained from the reported X-ray structures. The resulting homology model was refined using sequential NOE distance restraints, 13C chemical shift information and a conformational database.

The typical approach to homology modeling has relied heavily on sequence similarity between the new protein and target protein(s). The accuracy of the resulting homology model decreases significantly as the sequence homology between the structures drops below 40%. This is clearly problematic since a number of protein families have very divergent sequence homology (20% or less) while maintaining the same overall fold. Attempts have been made to use predictive methods to identify regions of secondary structure to generate alignments for homology modeling, but this approach is lacking since the validity of the resulting structure is dependent on both the combined accuracy of the structure prediction method and the homology modeling protocol. Nevertheless, the concept of using secondary structure alignment instead of sequence alignment to thread a homology model represents a better approach because of the higher information content.

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Pseudomonas putida Proline Utilization A, DNA-binding Domain (PutA)

Proline utilization A (PutA) is a multifunctional enzyme that allows Gram-negative bacteria, such as Escherichia coli and Pseudomonas putida, to utilize proline as a carbon and nitrogen source. PutA converts proline into glutamate in a two-step process that requires a proline dehydrogenase domain (PRODH) and a D1-pyrroline-5-carboxylate (P5C) dehydrogenase domain (P5CDH). PRODH and P5CDH are separate enzymes in Gram-positive bacteria, archea, and eukaryotes. Proline is first oxidized to P5C coupled with the reduction of the FAD cofactor by the PutA PRODH domain. The reduced FADH2 cofactor transfers the electrons to the electron transport chain system in the cytoplasmic membrane. The P5CDH domain then catalyzes the NAD+-dependant oxidation of P5C to glutamate.

PutA also functions as an autogenous transcriptional repressor by binding to multiple sites in the put regulatory region. Because the enzymatic activity of PutA requires PutA to be peripherally membrane bound, PutA function is regulated by proline-dependent switching of its intracellular location from the cytoplasm to the membrane. PutA can bind to the put control DNA in the absence (KD ~ 45 nM) and presence (KD ~ 100 nM) of proline suggesting changes in PutA-DNA binding affinity are not a major factor in functional switching. Rather, proline reduction of the FAD cofactor activates tight PutA-lipid binding (KD < 10 pM) leading to sequestration of PutA on the membrane. The tight membrane interaction thus prevents PutA from binding DNA. Previous studies have shown redox-dependent conformational changes occur in a linker region between the DNA binding and PRODH domains. Thus, a coupled conformational change involving the PRODH and DNA binding domains may be part of the redox mechanism by which PutA transfers from the cytoplasm to the membrane. The PutA DNA binding domain in E. coli is localized to the N-terminal 47 amino acids and is separated from the PRODH domain (261-612) by a flexible domain of unknown function (residues 141-260). This flexible domain incurs a significant conformational change upon proline binding, where membrane association of PutA is primarily driven by a hydrophobic interaction.

P. putida is a non-pathogenic organism with utility in bioremediation because of its metabolic diversity and ability to metabolize a wide-range of carbon sources. PutA from P. putida is a 1315 amino acid polypeptide and functions as a transcriptional repressor of the put regulon in a manner similar to E. coli PutA. The N-terminal residues 1-45 of PutA from P. putida (PpPutA45), was shown to be responsible for DNA binding and dimerization. The protein is a symmetrical homodimer (12 kDa) consisting of two ribbon-helix-helix (RHH) structures. PpPutA45 binds a 14 base-pair DNA oligomer (5'-GCGGTTGCACCTTT-3'). The antiparallel β-sheet that results from PpPutA45 dimerization serves as the DNA recognition binding site by inserting into the DNA major groove. The dimeric core of four α-helices provides a structural scaffold for the β-sheet from which residues Thr5, Gly7, and Lys9 make sequence specific contacts with the DNA. The structural model implies flexibility of Lys9 which can either make hydrogen bond contacts with guanine or thymine. The high sequence and structure conservation of the PutA RHH domain suggest interdomain interactions play an important role in the evolution of the protein.

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Human Regulators of G-Protein Signaling (RGS4)

An ubiquitous component of signal transduction pathways is a heterotrimeric guanine nucleotide-binding protein (G-protein) coupled to a cell surface receptor. G-proteins relay signals initiated by photons, odorants, and a number of hormones and neurotransmitters where a variety of diseases are caused by defects in G-protein activity. The structure of the G-protein is composed of an a-subunit (Ga) that is associated with both the intracellular carboxy terminal tail of a seven-helical transmembrane receptor and weakly bound to a dimer (Gbg) of a b-subunit tightly bound to a g-subunit. G-proteins transfer signals from more than 1000 receptors where various Ga subtypes regulate a variety of distinct downstream signaling pathways and the guanine nucleotide binding and GTPase function within the Ga domain regulates the activity of G-proteins.

The G-protein signaling process is typically initiated by the binding of an agonist to the cell surface receptor resulting in an induced conformational change in the G-protein. The G-protein structural change affects the guanine nucleotide affinity of Ga where it preferentially binds GTP and Mg2+ over GDP. Numerous x-ray structures for Gia1 during the various stages of the GTPase cycle has identified regions of induced conformational changes. In particular, the Ga guanine nucleotide binding site is composed of three distinct "switch" regions: residues V179-V185 in switch I, residues Q204-H213 in switch II and residues A235-N237 in switch III, which undergo conformational changes upon GTP hydrolysis. In the active Ga-GTP-Mg2+ complex, switch II and switch III regions become well ordered due to ionic interactions between the two switch regions where the conformational change in switch I is associated with binding Mg2+. The Ga surface that binds the Gbg dimer contains switch I and switch II regions. As a result of the formation of the Ga-GTP-Mg2+ complex, modifications in the structure of the three "switch" regions facilitate dissociation of Ga from Gbg. The released subunits are then available to interact with a variety of target proteins to elicit the desiyellow response. Termination of the signal results when the process is reversed by the hydrolysis of GTP bound to Ga. The re-association of Ga with Gbg then occurs which results in the inactivation of the G-protein. Therefore the duration of the G-protein signal is directly dependent on the GTPase activity of the Ga protein.

Regulators of G-protein signaling (RGS) affect the intensity and duration of the G-protein signal cascade by binding to the active Ga-GTP-Mg2+ complex and inducing a 50-fold increase in the rate of GTP hydrolysis. Conversely, RGS proteins have little to no affinity for the inactive Ga-GDP complex. Thus, RGS act as attenuators of the induced G-protein signal by increasing the rate of inactivation of the G-protein and termination of the signal. The RGS family contains more than 20 members where specificity for Ga subtypes has been demonstrated and is probably associated with subtle sequence differences. RGS proteins are widely expressed. At least one RGS protein is found in every organ where many tissues express multiple RGS proteins. Additionally, members of the RGS family have region-specific expression in the brain where RGS4 is perhaps the most widely distributed and highly expressed RGS subtype. The regulation of RGS expression suggest an adaptive response in the brain signal transduction pathway to compensate for desensitization and sensitization of G-protein-coupled receptor function since RGS expression has been correlated with a response to an induced seizure. In addition to response to neurotransmiters, RGS activity has been associated with a variety of cellular functions including: prolifferation, differentiation, membrane trafficking and embryonic development.

An X-ray structure of RGS4 bound to Gia1, site-directed mutagenous data, and biochemical studies suggests a potential mechanism for the RGS induced Ga hydrolysis of GTP. These results imply that RGS4 binds preferentially to the Ga-GTP-Mg2+ complex and stabilizes the transition state structure of the switch regions, stimulating the intrinsic GTPase activity. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding Gia1 as evident by the backbone atomic rms difference of 1.94 angstroms between the free and bound forms of RGS4. The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the Gia1 binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-Gia1 X-ray structure relative to the free RGS4 NMR structure resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-Gia1 X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for Gia1 on RGS4, where RGS4 residues D117, S118 and R121 interact with residue T182 from Gia1. The binding pocket for Gia1 on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the pre-formed binding site observed in the RGS4-Gia1 X-ray structure. This observation implies that the successful complex formation between RGS4 and Gia1 is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from Gia1. The observed changes for the free RGS4 NMR structure suggests a mechanism for its selectivity for the Ga-GTP-Mg2+ complex and a means to facilitate the GTPase cycle.

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Human Tumor Necrosis Factor Receptor-1 (TNFR-1) Death Domain

Activation of the tumor necrosis factor receptor-1 (TNFR-1) by the ligand TNF initiates two major intracellular signalling pathways that lead to the activation of the transcription factor NFkB and the induction of cell death, which also elicits a variety of biological responses, including antiviral activity, cytotoxicity, and modulation of gene expression. TNFR-1 through trimerization is induced by the binding of TNFa (cachectin) or TNFb (lymphotoxin a) trimers, inducing association of its intracellular death domain (DD). The trimerization of TNFR-1 alllows for the recruitment of an adaptor protein named TNFR-asociated death doman protein (TRADD) through death domain-death domain interaction. TRADD recruits the signaling molecules TNFR-associated factor-2 (TRAF-2) through interactions with the N-terminal domain, Fas-associated death domain protein (FADD) through death domain interactions, and the receptor interacting protein (RIP) through death domain interactions to form the TNFR-1 signaling complex. Based on similarities in their cystein-rich extracellular domains, TNFR-1 and TNFR-2 belong to a receptor superfamily, which besides a number of death inducing receptors, includes CD40 and the low-affinity nerve growth factor receptor. Although most cell types express both TNF receptors, TNFR-1 appears to play a predominant role in the induction of gene expression and induction of cell death by TNFa.

The death domain (DD) was originally described as a region of similarity within the intracellular portions of the TNFR-1 and Fas/Apo1 that is essential for the transduction of cytotoxic signals. It was subsequently shown that the death domain is a protein interaction motif involved in homo- and hetero-association. Besides being found in many proteins involved in signaling apoptosis, including receptors and downstream effectors, death domains are also present in other proteins that have different cellular functions, such as p75-NGFR, MyD 88, IRAK, MADD, N5, NFkB, DAP kinase, and ankyrins. The solution structure of FAS, FADDD and p75 neutrophin receptor death domains have recently been determined by NMR. They consist of a six antiparallel amphipathic a-helices packed into a globular structure. Two other domains called DED (death effector domain) and CARD (caspase recruitment domain) are structurally related to the death domains. Structural studies of death domains have been problematic due to the propensity to self-associate and form large molecular mass aggregates at physiological pH.

The low level of sequence conservation between the death domains probably reflects their role in diverse cellular functions. Additionally, the apparent absence of a conserved interaction surface suggests that death domains may associate by a variety of mechanisms. Indeed, the surface formed by α-helices 2 and 3 has been implicated in the homo- and hetero-association of the death domains of Fas and FADD. Similar observations have been made in the case of the interaction of the CARD domains of Apaf-1 and procaspase-9. Conversely, the dimerization of the Tube and Pelle death domains seem to rely on contacts between α-helices 4 and 5 of Pelle death domain with α-helix 6 and the unique C-terminal tail of the Tube death domain. The nature of the interaction also seems to be different between the death domain complexes. Electrostatic interactions are though to be a key component in the interaction between Fas death domain (FAS-DD) and FADD death domain (FADD-DD). Whereas, hydrogen bond contacts and Van der Waals interaction have been involved in the complex of Tube and Pelle death domains, as well as in the complex of Apaf-1 and procaspase-9 CARD domains. It is currently thought that the interactions between DEDs are hydrophobic.

NMR and mutational studies of human TNFR-1 death domain (TNFR-DD) and the mutant R347K TNFR1-DD exhibits the same general DD fold and that α2 and part of α3 and part of α4 are important for self-association and interaction with TRADD death domain (TRADD-DD).

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B. subtilis AHSA1 Protein (NESG SR211, YndB)

The Bacillus subtilis YndB protein is a protein of unknown biological function targeted for structural analysis by the Northeast Structural Genomics Consortium (NESG; http://www.nesg.org; NESG target: SR211). The YndB protein was originally assigned as a START domain protein based on the high sequence similarity to B. cereus BC4709 and B. halodurans BH1534, which were assigned to START domains based on common structural features. Instead, SCOP and Pfam databases have suggested that YndB belongs to the closely related AHSA1 subfamily. Also, YndB, BC4709, and BH1534 do not have the additional N-terminal β-strands and the additional α-helix that are characteristic of a START domain structure. Similarly, a BLASTP sequence alignment search indicates YndB is more appropriately assigned as a member of AHSA1. The functions for prokaryotic AHSA1 family members are typically classified as either a general stress protein or a conserved putative protein of unknown function.

The NMR structure for B. subtilis protein YndB indicates that the protein adopts a helix-grip fold and is clearly a member of the Bet v 1-like superfamily. The helix-grip fold consists of a β-sheet with two small and one long α-helix. The β-sheet is comprised of 5 strands instead of the normal 6. The YndB structure contains an apparent hydrophobic cavity between the long C-terminal &alpha-helix and the antiparallel β-sheet. Like other members of the Bet v 1-like superfamily the cavity suggests YndB binds to lipids, sterols, polyketide antibiotics, or other hydrophobic molecules as part of its biological function.

To further explore the potential functional annotation of YndB, an in silico screen against a ~18,500 lipid-like chemical library was conducted. The best binders identified from the in silico screen were from the three general lipid classes of flavones/flavonols, flavanones, and chalcones/hydroxychalcones. Representative compounds from all three classes were screened by NMR, where trans-chalcone, flavanone, flavone, and flavonol were all shown to bind in the YndB hydrophobic cavity with KD values of 1 μM, 32 μM, 62 μM, and 86 μM, respectively. A model for the YndB-chalcone complex was shown to be consistent with the binding of dilinoleoylphosphatidylcholine to human phosphatidylcholine transfer protein, a related START domain protein. The binding of chalcone and flavanone to a B. subtilis protein is an intriguing observation since these molecules are primarily found in plants as precursors to flavonoid molecules used for antimicrobial defense, flower pigmentation, absorption of harmful UV radiation, and signaling between plants and beneficial microbes. The sum of the data suggests an involvement of YndB with the stress response of B. subtilis to chalcone-like flavonoids released by plants due to a pathogen infection. The observed binding of chalcone-like molecules by YndB is likely related to the symbiotic relationship between B. subtilis and plants.

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E. coli Cell Division Protein (ZipA)

ZipA is essential for cell division and viability in E. coli. Experiments with the green fluorescent protein fusion of ZipA have demonstrated that only prior localization of FtsZ is required for localization of ZipA to midcell. Changes in the relative abundance of ZipA in the cell, either by depletion or over-expression, result in filamentation. The morphology of these filaments resembles those observed for cells in which FtsZ has been depleted. This is consistent with the observation that both ZipA and FtsZ are involved at the very early stage of cell division.

ZipA is a 328 amino acid protein that is composed of 5 regions or domains. The N-terminus is a highly hydrophobic region of approximately 25 amino acids which forms the transmembrane domain that anchors the protein to the cytoplasmic membrane. This is followed by a basic region (~ 23 amino acids), an acidic region (~17 amino acids), and a long proline-rich region. The C-terminal domain (residues 189-328) has been shown to be sufficient for binding to FtsZ and has several areas that are conserved among the seven ZipA sequences identified to date. Since ZipA is anchored to the cytoplasmic membrane while binding FtsZ, it has been speculated that the function of ZipA may be to link the membrane with the FtsZ rings, to stabilize or organize the FtsZ rings, or to link invagination of the membrane to constriction of the FtsZ ring during septation. Since changes in the relative abundance of ZipA in the cell result in filamentation, disruption of the ZipA-FtsZ interaction would likely disrupt cell division and cause cell lysis, suggesting that the ZipA-FtsZ interaction may be a viable therapeutic target for drug development.

The NMR solution structure of ZipA185-328 is comprised of three α-helices and a β-sheet consisting of six anti-parallel β-strands where the α-helices and the β-sheet form surfaces directly opposite each other. A C-terminal peptide from FtsZ has been shown to bind ZipA185-328 in a hydrophobic channel formed by the β-sheet providing insight into the ZipA-FtsZ interaction. An unexpected similarity between the ZipA185-328 fold and the split β-α-β fold observed in many RNA binding proteins may further our understanding of the critical ZipA-FtsZ interaction.

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