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| ==MgtC: A Virulence Factor From ''Mycobacterium tuberculosis''== | | = DgcZ from ''E. coli'' = |
| <StructureSection load='2lqj' size='340' side='right' caption='C-terminal Domain of Mg2+ transport P-type ATPase C (PDB: [http://www.rcsb.org/pdb/explore.do?structureId=2lqj 2LQJ])' scene='69/698113/Rainbow-colored_spectrum/2'>
| | [[Image:Zn_Binding_Site_DgcZ.png|250 px|left|thumb|Zn Binding Site DgcZ. The Cys52 reside is not the N-terminal residue, but the rest of the 𝝰helix 2 was not successfully crystallized.]] |
| ==Introduction==
| | <Structure load='4h54' size='350' frame='true' align='right' caption='4h54' scene='Insert optional scene name here' /> |
| [http://en.wikipedia.org/wiki/Tuberculosis Tuberculosis], caused by ''[http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis Mycobacterium tuberculosis]'', is a [http://en.wikipedia.org/wiki/Respiratory_tract_infection respiratory infection] still prevalent throughout the world. During the last decade, the emergence of [http://en.wikipedia.org/wiki/Multiple_drug_resistance multi-drug resistant] strains of ''M. tuberculosis'' has given rise to the need for the development of new [http://en.wikipedia.org/wiki/Antibiotics antibiotics] in order to combat the infection<ref>Singh, G.; Singh, G.; Jadeja, D.; Kaur, J. Lipid hydrolyzing enzymes in virulence: Mycobacterium tuberculosis as a model system. Critical Reviews in Microbiology 2010, 36(3): 259-269. DOI: 10.3109/1040841X.2010.482923.</ref>. In order to develop an efficacious antibiotic, the drug must be able to target a unique aspect of the bacteria, such as a protein, that is critical for its full virulence and survival. MgtC, an [http://en.wikipedia.org/wiki/Integral_membrane_protein integral protein] embedded in the extracellular membrane of ''M. tuberculosis'', has recently been hypothesized as a novel drug target to resolve tuberculosis infections. The targeting of MgtC was a result of observing that upon deletion of the protein from ''M. tuberculosis'', the bacteria are no longer able to survive due to inhibition of [http://en.wiktionary.org/wiki/intramacrophage intramacrophage] growth. <ref name="mgtc">Yang, Y.; Labesse, G.; Carrere-Kremer, S.; Esteves, K.; Kremer, L.; Cohen-Gonsaud, M.; Blanc-Potard, A. The C-terminal domain of the virulence factor mgtc is a divergent act domain. J Bacteriol. 2012, 194(22): 6255-6263. DOI: 10.1128/JB.01424-12.</ref>. | |
| == Structure == | |
| Based on its [http://en.wikipedia.org/wiki/Protein_tertiary_structure tertiary structure], this protein has been placed into a larger group of proteins known as the [http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=120498 MgtC superfamily]. The overall structure of MgtC is constituted by two [http://en.wikipedia.org/wiki/Protein_domain domains]: an N-terminal domain and a C-terminal domain. Each of these domains have striking similarities and differences with other MgtC-like proteins.<ref name="mgtc"/>
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| ===N-terminal Domain=== | | == Biological Function == |
| The N-terminal domain of MgtC is highly-conserved between [http://en.wiktionary.org/wiki/orthologue orthologs] of the MgtC [http://en.wikipedia.org/wiki/Protein_superfamily super family]. This domain is largely hydrophobic and serves as the main component of MgtC that allows its embedment in the extracellular membrane. While this domain is highly conserved among orthologs, a [http://en.wikipedia.org/wiki/Crystal_structure crystal structure] is not yet available, but the sequence available has determined it to be largely hydrophobic. <ref name="mgtc"/>
| | Diguanylate cyclases, class 2 transferase enzymes, catalyze the production of cyclic dimeric-guanosine monophosphate (c-di-GMP), important to signal transduction as a second messenger. Signal transduction is the process of sending signals through cells to promote responses most commonly through phosphorylation or dephosphorylation events. Enzyme DgcZ from ''E. coli'' acts a catalyst to synthesize cyclic di-GMP from two substrate guanosine triphosphate (GTP) molecules to aid in communication of signals throughout the bacteria. C-di-GMP is a second messenger in the production of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a polysaccharide required for ''E. coli'' biofilm production. This biofilm allows ''E. coli'' to adhere to extracellular surfaces. The DgcZ protein has C2 symmetry composed of two domains: the catalytic glycine-glycine-glutamate-glutamate-phenylalanine (GGEEF) domain responsible for synthesizing c-di-GMP and the regulatory chemoreceptor zinc binding (CZB) domain comprising two zinc binding sites. DgcZ binds zinc with sub-femtomolar affinity. When zinc is bound, the CZB and GGEEF domains adopt conformations that inhibit DgcZ function. |
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| ===C-terminal Domain=== | | == Structural Overview == |
| This domain of MgtC, in contrast, is highly variable in comparison to several orthologs, as presented by Yang ''et al''. However, it is this <scene name='69/698113/Secondary_structured_coloring/2'>tertiary structure</scene> containing two α-helices and four anti-parallel β-sheets that is incredibly indicative of the MgtC super family. Through a sequence alignment of five known functional MgtC orthologs from [http://en.wikipedia.org/wiki/Pathogen pathogens] that survive inside [http://en.wikipedia.org/wiki/Macrophage macrophages] (''M. tuberculosis, [http://en.wikipedia.org/wiki/Brucella_melitensis B. melitensis], [http://en.wikipedia.org/wiki/Burkholderia_cenocepacia B. cenocepacia], [http://en.wikipedia.org/wiki/Yersinia_pestis Y. pestis],'' and ''[http://en.wikipedia.org/wiki/Salmonella_enterica_subsp._enterica S. Typhimurium]''), seven strictly conserved residues were found to be scattered along the whole sequence of the relatively hydrophilic and soluble C-terminal domain. <ref name="mgtc"/>
| | Enzyme DgcZ has been co-crystallized with Zinc conforming it to its inactivated conformation. The CZB domain is common to many bacterial lineages, appearing most commonly in bacterial chemoreceptors involved in <span class="plainlinks">[https://en.wikipedia.org/wiki/Chemotaxis chemotaxis]</span>. The second most common group of CZB domains is that of DgcZ homologs. [1]. The domain has an important role in signal transduction of bacteria[1]. 30 small bacterial proteins of family PRK0984 from differing strands of ''E. coli'' contain a CZB domain N-terminal to a GGDEF domain[1]. The GGEEF domain of DgcZ is common to this family of enzymes containing the GGDEF domain. ''E. coli'' DgcZ is a protein made of two domains each of which is a symmetric homodimer. The GGEEF domain is catalytic in that it contains the active sites used for cyclizing GTP into c-di-GMP. The CZB domain is used for ligand-mediated regulation of c-di-GMP production. Zinc binds as an allosteric inhibitor in coordination with four residues to shift the protein into an inactive conformation. |
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| A large hydrophobic core has conserved residues <scene name='69/698113/Colored_core_residues/6'>Cysteine-155, Arginine-164, Glutamine-160, and Alanine-195</scene>. | | ===Catalytic GGEEF Domain=== |
| | The GGEEF domain of DgcZ is part of the GGDEF family of proteins that includes a conserved sequence, GG[DE][DE]F[2].The GGEEF domain is a homodimer consisting of a central five-stranded β-sheet surrounded by five α-helices. Each dimer contains an active half-site that, when combined together in a productive conformation, form the entire active site. Each half-site binds one GTP molecule. The guanyl base forms hydrogen bonds with Asp-173 and Asn-182 to hold it in the active site. A Mg<sup>2+</sup> ion stabilizes the negative charges on the phosphate groups. When in the productive conformation, each GTP is held in close proximity with the α-phosphate groups overlapping C3 of the ribose. This conformation allows the α-phospate of one GTP to react with the alcohol group on C3 of the ribose of the other GTP, resulting in a cyclization of the two molecules into c-di-GMP. The ribose of each guanosine triphosphate, and subsequent product c-di-GMP riboses, are held only loosely by the enzyme, while the phosphate groups are not bound at all. |
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| [[Image:Final_Final_Core_Logo.PNG |625× 116px|thumb|left|Four strictly conserved residues of five known functional MgtC orthologs of the soluble C-terminal domain.
| | ===Mechanism of Action=== |
| The figure was prepared using WebLogo. (http://weblogo.berkeley.edu/)]]
| | Diguanylate cyclases only function efficiently as dimers, to bind both GGDEF domains holding the substrates. The presence of Zinc disrupts the ability of the two domains to overlap. |
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| | 1. The enzyme coordinates the substrate GTP to allow for deprotonation of the C3 -OH groups of the ribose. The negatively charged Oxygens on the phosphate groups of GTP are stabilized by Mg<sup>2+</sup> ions. |
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| | 2. The deprotonated Oxygen then acts as a nucleophile to attack the 𝝰phosphate of GTP. |
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| | 3. The β and γ phosphates of GTP are kicked off to form c-di-GMP. |
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| | ===CZB Domain=== |
| | The CZB domain is responsible for regulating the function of DgcZ. The domain contains the allosteric binding site of the enzyme with cooperative binding. Four residues bind zinc with a high affinity even at 10<sup>-16M</sup> concentrations. Due to the tightness of Zinc binding, the enzyme has not yet been crystallized in the active conformation without the presence of Zinc metal inhibitor. |
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| | [[Image:Zinc coordination DgcZ.png|250 px|left|thumb|Zn Coordination to amino acid residues on three of the four 𝝰 helices of DgcZ]] |
| | === Zinc Binding Site === |
| | Most cells possess efficient Zinc uptake systems, as Zinc is a reactive Lewis Acid. Zinc binds incredibly tightly to this enzyme at subfemtomolar concentrations. The Zinc co-purified with the protein.Zinc allosterically inhibits the activity of enzyme DgcZ through two allosteric binding sites located on the CZB domain. The inhibition prevents regulation of GGDEF domain function, the location of the active site. The CZB domain is folded into four anti-parallel α-helices as a 2-fold symmetric homodimer, with the N-terminus on the helix 𝝰4. The allosteric binding site includes amino acids, H22 of 𝝰1, C52 of 𝝰2, and H79 and H83 of 𝝰3, that span three of the four alpha helices of the CZB domain coordinating the Zinc residue in a tetrahedral fashion. Zahringer et al. mutated Cys52 to Ala through <span class="plainlinks">[http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/5/3/18.html site-directed mutagenesis]</span>, resulting in a lack of coordination on α2. The cysteine residue is not essential for Zinc binding, as Zinc still coordinates to the three His residues with the Cys52Ala mutation, but α2 is free to move and expose the Zinc binding pocket. This exposure was found to lower the protein's affinity for zinc, as the mutation of cysteine to alanine increased the activity of the DgcZ. Using EDTA, Zinc can be removed from the CZB domain. The zinc has higher affinity for EDTA than CZB when EDTA concentration is higher than the concentration of DgcZ. When not coordinated to zinc, the CZB domain adopts a conformation that straightens the 𝝰1 helix shifts, shifting hydrophobic residues on the α-helices into the center and the GGEEF domain into its productive conformation, increasing activity of DgcZ. Activity increases without Zinc due to activation of poly-GlcNAc production and biofilm formation, and maximal cyclic di-GMP production. |
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| | | This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. |
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| The opposite side of the protein has a small cluster of conserved residues <scene name='69/698113/Conserved_surface_residues/6'>Tyrosine-149, Glutamine-208, and Tryptophan-225</scene>.
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| [[Image:Final_Surface_Web_Logo.PNG |625× 121px|thumb|left|Four strictly conserved residues of five known functional MgtC orthologs of the soluble C-terminal domain. The figure was prepared using WebLogo. (http://weblogo.berkeley.edu/)]]
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| Additionally, there is a crystal structure available for this domain. When comparing the crystal structure of the C-terminal domain to other protein structures, there are striking similarities between this domain and a class of proteins known as [http://en.wikipedia.org/wiki/ACT_domain ACT domains]. <ref name="mgtc"/>
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| ==Function==
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| Collectively, because there is not a crystal structure available for the entire protein and the high variability of the C-terminal domain, it has been difficult to characterize the biochemical function performed by MgtC within ''M. tuberculosis''. Several roles have been proposed, including magnesium uptake, the binding of amino acids and metals, as well as facilitating dimerization with various proteins. <ref name="mgtc"/>
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| ===Magnesium Transport===
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| A role for MgtC as a [http://en.wikipedia.org/wiki/Magnesium_transporter magnesium transporter] has been debated since its discovery. Several publications have produced data indicating that this protein is critical for the uptake of magnesium in magnesium-deprived medium, while other literature has shown that this protein plays an insignificant role in this process. <ref name="mgtc"/> <ref>Blanc-Potard, A.B.; Lafay, B. MgtC as a horizontally-acquired virulence factor of intracellular bacterial pathogens : evidence from molecular phylogeny and comparative genomics. J Mol Evol. 2003, 57(4): 479-86. DOI: 10.1007/s00239-003-2496-4 </ref> <ref>Belon, C.; Gannoun-Zaki, L.; Lutfalla, G.; Kremer, L.; Blanc-Potard, A.B. Mycobacterium marinum mgtc plays a role in phagocytosis but is dispensable for intracellular multiplication. Plos One 2014, 1-23. DOI: 10.1371/journal.pone.0116052. </ref>
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| Support for a role in magnesium transport is supported by: 1) Mutants of MgtC are unable to survive in low-magnesium environment; 2) Expression of the gene encoding for MgtC is highly-induced in low magnesium environment; 3) Genes adjacent to the MgtC gene encode for known magnesium transporters.
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| Very recent evidence against MgtC playing a role in magnesium transport showed that [http://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction RT-PCR] experiments gave consistent levels of MgtC expression despite changes in the concentration of extracellular magnesium. <ref name="mgtc"/>
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| ===Potential for Binding Amino Acids===
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| The exploration of this role for MgtC was first considered because of the ACT domain-like structure of the C-terminal domain.
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| ACT domains commonly bind small amino acids within the cell as a form of [http://geneontology.org/page/regulation regulation]. Yang ''et al''. showed that the structure of the C-terminal domain overlaps significantly with the structure of [http://proteopedia.org/wiki/index.php/1psd SerA] (PDB: [http://www.rcsb.org/pdb/explore/explore.do?structureId=1psd 1PSD]), a known amino acid-binding ACT domain from ''[http://www.cdc.gov/ecoli/ E. coli]''. '''Figure 1A''' shows the overlap of these two proteins; the cyan protein represents MgtC and the orange protein represents SerA. However, the glycine that is critical for the binding of amino acids in these ACT domains has been substituted in MgtC with a <scene name='69/698113/Sub_residues_of_sera/2'>tyrosine</scene>, likely abolishing any potential amino acid binding activity <ref name="mgtc"/>
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| ===Potential for Chelation===
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| As with the potential for binding amino acids, this role was also explored because of the structural similarity of the C-terminal domain with ACT domains, as ACT domains also serve as excellent [http://en.wikipedia.org/wiki/Chelation chelators] to sequester cations within the cell. Yang ''et al''. also compared the structure of the C-terminal domain of MgtC with an ACT domain of a known chelator, [http://proteopedia.org/wiki/index.php/3lgh NikR] (PDB: [http://www.rcsb.org/pdb/explore/explore.do?structureId=3LGH 3LGH]). These structures overlapped quite well, indicating that MgtC may serve as a chelator. '''Figure 1B''' highlights the significant overlap between these residues; the cyan protein represents MgtC and the orange protein represents NikR. However, the two histidine residues and the cysteine residue present in NikR that serve as the chelating residues are modified to <scene name='69/698113/Sub_residues_of_chelat/3'>threonine, proline, and isoleucine</scene> respectively. These substitutions likely prevent any chelating activity by MgtC. <ref name="mgtc"/>
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| [[Image:Combined_overlaps.png |458 x 210 px|thumb|center|'''Figure 1. Overlap of the C-terminal Domain of MgtC with ACT domains of known function.''' 1A shows the significant overlap of the C-terminal of MgtC with SerA, an ACT domain that has been established to bind amino acids. 1B shows the overlap of the C-terminal domain of MgtC with NikR, a known chelating ACT domain.]]
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| ===Role in Dimerization===
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| The potential for [http://en.wikipedia.org/wiki/Protein_dimer dimerization] was another aspect of MgtC studied to see if this protein forms complexes with proteins of known function. A [http://subtiwiki.uni-goettingen.de/wiki/index.php/BACTH Bacterial Two-Hybrid (BACTH)] assay was performed to study the potential for the entire protein to dimerize with itself and the potential for individual domains to dimerize. The results of this assay showed that the entire MgtC protein likely dimerizes, but the individual domains do not. This dimerization could serve as a critical component to the biochemical function of MgtC, although the exact implications have not yet been discerned <ref name="mgtc"/>. Frantz ''et al''. proposed a role for MgtC to form dimers with [http://proteopedia.org/wiki/index.php/2mc7 MgtR] (PDB: [http://www.rcsb.org/pdb/explore/explore.do?structureId=2MC7 2MC7]), a protein that serves to promote the degradation of MgtC.<ref name="mgtr">Jean-Francois, F.L.; Dai, J.; Yu, L. ; Myrick, A. ; Rubin, E. ; ''et al''. Binding of mgtr, a salmonella transmembrane regulatory peptide, to mgtc, a mycobacterium tuberculosis virulence factor: a structural study. DOI:10.1016/j.jmb.2013.10.014</ref> This has huge implications in the overall clinical relevance of how MgtC could be targeted to develop new-generation antibiotics.
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| ==Clinical Relevance ==
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| The development of an antibiotic which targets and inhibits MgtC could come from exploitation and enhancement of the process which promotes its degradation within ''Mycobacterium tuberculosis.'' MgtR, a hydrophobic peptide, promotes the degradation of MgtC upon high expression within the bacteria.<ref name="mgtr"/> As previously stated, inadequate levels of MgtC within ''M. tuberculosis'' results in an inability to grow and survive. <ref name="mgtr"/> It is quite reasonable that analogues of MgtR could be developed, injected ([http://en.wikipedia.org/wiki/Subcutaneous_injection subcutaneously]) into infected patients, and resolve the tuberculosis infection by promoting degradation of MgtC and impairing growth of ''M. tuberculosis.''
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| ==Future Work==
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| [[Image:Aligned 3.png|210 px|thumb|right|'''Figure 2. Overlap of MgtC C-terminal domain with the ACT domain of a GTP pyrophosphokinase.''' This figure demonstrates the significant overlap between the C-terminal domain of MgtC and the ACT domain of a GTP pyrophosphokinase.]]
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| Since so little is known about MgtC, future work should involve both crystallizing the entire MgtC protein and characterizing its biochemical function. Because the sequence of amino acids in a protein dictates structure, and structure typically determines the protein's function, further sequencing and structural analysis should be performed with MgtC to discern its function. Shown in '''Figure 2''' is an overlap of MgtC (cyan) with the ACT domain of a [http://en.wikipedia.org/wiki/GTP_diphosphokinase GTP pyrophosphokinase] (PDB: [http://www.rcsb.org/pdb/explore/explore.do?structureId=2kO1 2KO1]) shown in orange. This overlap shows even more extensive similarity than the aforementioned SerA and NikR ACT domains. Structural similarity analysis could aid in resolving the biochemical function of MgtC.
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| </StructureSection> | | </StructureSection> |
| == References == | | == References == |
| <references/> | | <Jenny Draper, K. Karplus, K. Ottemann. Identification of a Chemoreceptor Zinc-Binding Domain Common to Cytoplasmic Bacterial Chemoreceptors. Journal of Bacteriology. Vol. 193, No. 17. 4338-4345. (2011).> |
| | <Carmen Chan, R. Paul, D. Samoray, N. Amiot, B. Giese, U. Jenal, T. Schirmer. Structural basis of activity and allosteric control of diguanylate cyclases. PNAS. Vol 101. No. 49 17084-17089. (2004).> |