User:Jamie Abbott/Sandbox2

== Histidyl-tRNA Synthetase ==

'''Histidyl tRNA Synthetase (HisRS)''' is a 94kD <scene name='User:Jamie_Abbott/Sandbox2/Hisrsdimer/2'>homodimer</scene> that belongs to the class II of aminoacyl-tRNA synthetases (aaRS). [http://www.pdb.org/pdb/101/motm.do?momID=16 Aminoacyl-tRNA synthetases] Aminoacyl-tRNA synthetases have been partitioned into two classes, containing 10 members, on the basis of sequence comparisons<ref name="Eriani">PMID: 2203971</ref>. Class I and Class II differ mainly with respect to the topology of the catalytic fold and site of esterification on cognate tRNA<ref name="Eriani" />. Class II enzymes have a <scene name='User:Jamie_Abbott/Sandbox2/Catalytic_domain/1'>catalytic domain</scene> composed of anti-parallel β-sheets and α-helices (residues 1-325). Additionally, class II enzymes can be further divided into three subgroups: class IIa, distinguished by an N-terminal catalytic domain and C-terminal accessory domain (later shown to be anticodon binding domain); class IIb, whose anticodon binding domain is located on the N-terminal side of the fold; and class IIc, encompassing the tetrameric PheRS and GlyRS class II synthetases.<ref name="Cusack91">PMID: 1852601</ref>

<StructureSection load='1KMM' size='500' side='right' caption='Structure of Escherichia coli Histidyl-tRNA Synthetase (PDB entry [[1KMM]])' scene=''>Class II aminoacyl-tRNA synthetases aminoacylate the 3'OH of their cognate tRNAs.

'''Histidine Binding Pocket'''

The active site to HisRS contains a histidine binding pocket <scene name='User:Jamie_Abbott/Sandbox2/Histidine_binding_residues/1'>histidine binding pocket</scene> composed of highly conserved residues found in distinct sequences motifs. First, the LV/AAGGGLDYY loop (or <scene name='User:Jamie_Abbott/Sandbox2/Hisa_loop/1'>HisA Loop</scene> ) forms one wall of the binding pocket. This HisA loop is highly conserved and extends over a part of the active site<ref name="aaRSbk">Francklyn, C., and Arnez, J.G. (2004) in ''Aminoacyl-tRNA Synthetases'' (Ibba, M.,Francklyn, C.,Cusack, S.. Eds.) [http://www.landesbioscience.com/books/iu/id/810/?nocache=145477703 Landes Publishing, Austin, TX]</ref>. Second, the glycine-rich β-strand (sequence AGGRYDGL preceding <scene name='User:Jamie_Abbott/Sandbox2/Motif_iii/4'>motif III</scene>) comprises the histidine binding pocket floor and wall. Finally, conserved side chains that make direct contact with histidine are Glu83 and Gly127 (<scene name='User:Jamie_Abbott/Sandbox2/Motif_ii/2'>motif II</scene>), which contact the α-amino and α-carbonyl functional groups, respectively, and Glu131 (motif II) and Tyr264, which make hydrogen bonds to the Nδ and Nε, respectively, of the imidazole ring<ref name="aaRSbk" />.

'''Adenosine Triphosphate Binding'''

Many interactions are required to prepare ATP for attack by a bound histidine molecule and encourage the magnesium pyrophosphate moiety to act as a leaving group. Residues in the β strands and the loop portion of motif 2 are important in ATP contacts for HisRS<ref name="Arnez97">PMID: 9207058</ref>. Generally, residues involved in ATP binding are among the most highly conserved in the HisRS family and for the most part shared by all members in class II. The π-stacking interaction between the adenine ring of ATP and <scene name='User:Jamie_Abbott/Sandbox2/Atp_phe125_pi-interaction/1'>Phe125</scene> provides specificity in the binding of ATP. The recognition of the N6 amino group of ATP involves the main chain carbonyl of Tyr122. The ATP ribose 2’ OH forms an additional contact with HisRS by hydrogen bonding with the main chain carbonyl of Thr281. Furthermore, conserved residues Arg113 and Glu115 stabilize the triphosphate group of ATP in a position such that it points back towards the adenine base. This <scene name='User:Jamie_Abbott/Sandbox2/Atp_fishhook/1'>“fishhook”</scene> conformation of ATP is evidently unique to class II aaRS<ref name="Arnez97-2">PMID: 9204708</ref>.

The α phosphate of ATP interacts with conserved residue Arg113. The β and γ phosphates are neutralized by two coordinated magnesium ions that are positioned by water molecules and conserved Glu115<ref name="aaRSbk" />. Also, the γ phosphate forms additional interactions with conserved Arg121 and Arg311.<scene name='User:Jamie_Abbott/Sandbox2/Atp_binding_residues/1'>ATP binding residues</scene></StructureSection>

<scene name='User:Jamie_Abbott/Sandbox2/Hisrsdimer_to_monomer/2'>Monomer</scene>

<scene name='User:Jamie_Abbott/Sandbox2/Motif_i/3'>Motif I</scene>

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== Mechanism ==

[[Adenylation.jpg |center|thumb|500px| '''Adenylation reaction catalyzed by HisRS''']]

[[Aminoacylation.jpg|center|thumb|500px| '''Aminoacylation reaction catalyzed by HisRS''']]

=== Electrophilic Catalysis ===

The HisRS active site contains a highly conserved residue, Arg259, takes part in electrophilic catalysis for the adenylation reaction. First, as Arg259 is positioned on the HisA loop serves to fix the α-carboxylate group of the histidine substrate as the attacking nucleophile<ref>PMID: 9715912</ref>. Second, the guanidinium group of Arg259 is positioned approximately 3Å from the α-phosphate of ATP where it serves as the electrophilic catalyst. Arg113 as well as Arg259 are arranged to interact with α-phosphate of ATP and thereby stabilize negative charge developed on the non-bridging oxygens during the transition state aarsbk. Evidence for Arg259 playing a critical role in catalysis is observed in a two or three log decrease in activity when substituted with a histidine <ref name="Arnez97" /> or other amino acids<ref>PMID: 9266856</ref>. Arg259 also interacts with the phenolic OH of Tyr264, which in turn donates a hydrogen bond to the Nδ of the histidine substrate<ref name="aaRSbk" />. Utilizing Arg259 for catalysis is unique to HisRS as other class II aaRS enzymes, AspRS<ref>PMID: 7966328</ref> and SerRS<ref>PMID: 7613865</ref>, use a divalent magnesium metal ion to coordinate the α-phosphate of ATP and serve as an electrophilic catalysis.

=== Substrate Assisted Catalysis ===

The second reaction carried out by HisRS, aminoacylation, requires the decomposition of a mixed anhydride (the <scene name='User:Jamie_Abbott/Sandbox2/Histidyl-adenylate/1'>histidyl-adenylate</scene>) to form an aminoacyl ester on the 3’OH of tRNAHis. It was initially hypothesized that Glu83, acting as a general base, would improve the rate of this reaction. However, while Glu83 is in a favorable position in the active site to function as a base it is also situated to neutralize the α-amino group of the histidine substrate. Thus, mutational analysis of Glu83<ref name ="GUTH05">PMID: 15751955</ref> suggests that it does not act as a base but forms a salt bridge with the α-amino group of histidine, neutralizing it’s charge, and satisfying a critical electrostatic interaction.

The mechanism for transfer of the aminoacyl moiety from the aminoacyl-adenylate, generated in the adenylation reaction, onto it’s cognate tRNA is most easily explained by '''substrate assisted catalysis (SAC)'''. Substrate assisted catalysis preformed by HisRS is described as the occurrence of bond formation between the 3’OH of tRNAHis and the α-carboxylate carbon of the aminoacyl adenylate prior to the cleavage of the bond joining the α-carboxylate carbon to the axial oxygen of the α-phosphate. This concerted SAC mechanism, rationalizes the considerable pKa difference between the 3’OH of tRNA (pKa =16) and the nonbridging Sp oxygen (pKa= -1)<ref name="GUTH05" />. Therefore, as the bond between the tRNAHis nucleophile and the α-carboxylate is formed, the pKa for the 3’OH would be expected to drop sharply and the pKa of the nonbridging oxygen would tend to rise as the bond to the α-carboxylate lengthens and breaks<ref name="GUTH05" />.

== Histidine tRNA Recognition ==

[[Image:TRNAHis2.jpg |thumb|right|upright=2.0|400px|'''Clover leaf structure of histidine tRNA from E.coli''' key recognition elements are shown in red]]

The accuracy of protein synthesis is dependent upon the ability of aminoacyl-tRNA synthetases to specifically recognize the cognate tRNA and attach the appropriate amino acid. The identity nucleotides that define tRNA isoacceptor systems are primarily concentrated in the anticodons and acceptor stems of [http://proteopedia.org/wiki/index.php/TRNA tRNAs], providing functional groups that can be accessed by specificity-determining side chains on the enzymes (8422978, 1857417). Although, tRNA identity can also emerge from the presence of modified bases (3054566). Key identity elements on ''E. coli'' histidine tRNA include the 5’ phosphate, G-1:C73 base pair in the acceptor stem and the GUG anticodon. Mutations of these identity elements diminishes aminoacylation ''in vitro''<ref>PMID: 10747795</ref><ref>PMID: 2678006</ref><ref>PMID: 8643360</ref>. Residues throught to be involved in the recognition of these identity elements include; Arg123, Arg116, and Gln118. Substitution in Arg123 as a putative contact to the 5’phosphate, produced a 200 fold decrease in aminoacyl-transfer<ref name ="GUTH07">PMID: 17317626</ref>. Similar kinetic defects in aminoacyl-transfer were also observed for Arg116 and Gln118 <ref name="GUTH07" />.

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== Evolutionary Conservation ==

=== Structural Homology ===

== 3D Structures of Histidyl-tRNA Synthetase ==

'''Bacteria'''

[http://www.pdb.org/pdb/explore/explore.do?structureId=1KMN 1KMN]

[http://www.pdb.org/pdb/explore/explore.do?structureId=1KMM 1KMM]

[http://www.pdb.org/pdb/explore/explore.do?structureId=1HTT 1HTT]

[http://www.pdb.org/pdb/explore/explore.do?structureId=2EL9 2EL9]

'''Eukaryota'''

[http://www.pdb.org/pdb/explore/explore.do?structureId=3LC0 3LCO]

[http://www.pdb.org/pdb/explore/explore.do?structureId=3HRK 3HRK]

'''Archara'''

[http://www.pdb.org/pdb/explore/explore.do?structureId=1WU7 1WU7]

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== References ==

<references/>