Journal:Acta Cryst F:S2053230X18016217
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The crystal structure of ''M. tuberculosis'' HtrA (ΔTM HtrA) that was determined at 1.83 Å resolution. We note that this enzyme exhibits both monomeric as well as trimeric forms in solution. The structure reveals a conformation that would require minor structural alterations for proteolytic activity. Structural features thus suggest that ''M. tuberculosis'' HtrA is a regulated protease as opposed to the two other paralogues, PepD and PepA. This essential enzyme is thus likely to be involved in specific signal transduction role as opposed to housekeeping in the recognition and degradation of partially folded or misfolded proteins. | The crystal structure of ''M. tuberculosis'' HtrA (ΔTM HtrA) that was determined at 1.83 Å resolution. We note that this enzyme exhibits both monomeric as well as trimeric forms in solution. The structure reveals a conformation that would require minor structural alterations for proteolytic activity. Structural features thus suggest that ''M. tuberculosis'' HtrA is a regulated protease as opposed to the two other paralogues, PepD and PepA. This essential enzyme is thus likely to be involved in specific signal transduction role as opposed to housekeeping in the recognition and degradation of partially folded or misfolded proteins. | ||
| - | The <scene name='80/801748/Cv/9'>crystal structure of ΔTM HtrA</scene> was determined at a resolution of 1.83Å (PDB ID: [[6ieo]]). In this crystal form, there is one molecule of HtrA in the asymmetric unit. The structure of the periplasmic domain reveals one <scene name='80/801748/Cv/10'>trypsin-like protease domain</scene> (<span style="color:royalblue;background-color:black;font-weight:bold;">226-436; colored in royalblue</span>) flexibly tethered to the <scene name='80/801748/Cv/11'>PDZ domain</scene> (<span style="color:gold;background-color:black;font-weight:bold;">443-528; in gold</span>) at the C-terminal end. The <scene name='80/801748/Cv/12'>protease domain consists of two β-barrels</scene> ({{Template:ColorKey_Helix}},{{Template:ColorKey_Strand}}, {{Template:ColorKey_Loop}}, {{Template:ColorKey_Turn}}) referred to as the N-terminal and C-terminal β-barrel. While the N-terminal β-barrel contains the active site residues His270 and Asp306, the C-terminal β-barrel has Ser387 from <scene name='80/801748/Cv/13'>the catalytic triad</scene>. The substantial structural conservation across HtrA enzymes suggests a similar reaction mechanism as evident from the positive charge cavity (the oxyanion hole) which helps in stabilization of tetrahedral intermediate during the acylation step of catalysis. The side chain of the active site serine, Ser387, could be modelled in two alternate conformations with an occupancy of 0.53 and 0.47. Of note, that N<sub>δ1</sub> (His270) and O<sub>δ1</sub>/O<sub>δ2</sub> (Asp306) are within <scene name='80/801748/Cv/14'>hydrogen bonding distance</scene> (2.6Å /3.2Å). On the other hand, the orientation of active site histidine places N<sub>ε2</sub> of His270 <scene name='80/801748/Cv/15'>significantly away</scene> from the O<sub>ϒ</sub> of Ser387. The orientation of H270 (N<sub>ε2</sub>) and Ser387 (O<sub>γ</sub>) (separated by ''ca'' 8.0Å) suggests that this crystal structure represents an inactive conformation. The PDZ domain is linked to the protease domain by a twelve residue long polypeptide segment. Based on extensive analysis of ''E. coli'' DegS, the L1 (157-167) and L3 (141-148) loops are essential for regulation of protease activity whereas the L2 loop governs substrate specificity.<ref name="Hasenbein">PMID:20184896</ref> These loops connecting helices or strands in protease domain are highlighted (green). The movement of L3 loop away from PDZ domain has been shown to shift the equilibrium from the inactive to active state of DegS upon peptide binding to PDZ domain.<ref name="Sohn">PMID:19836340</ref> In the ''M. tuberculosis'' HtrA structure, | + | The <scene name='80/801748/Cv/9'>crystal structure of ΔTM HtrA</scene> was determined at a resolution of 1.83Å (PDB ID: [[6ieo]]). In this crystal form, there is one molecule of HtrA in the asymmetric unit. The structure of the periplasmic domain reveals one <scene name='80/801748/Cv/10'>trypsin-like protease domain</scene> (<span style="color:royalblue;background-color:black;font-weight:bold;">226-436; colored in royalblue</span>) flexibly tethered to the <scene name='80/801748/Cv/11'>PDZ domain</scene> (<span style="color:gold;background-color:black;font-weight:bold;">443-528; in gold</span>) at the C-terminal end. The <scene name='80/801748/Cv/12'>protease domain consists of two β-barrels</scene> ({{Template:ColorKey_Helix}},{{Template:ColorKey_Strand}}, {{Template:ColorKey_Loop}}, {{Template:ColorKey_Turn}}) referred to as the N-terminal and C-terminal β-barrel. While the N-terminal β-barrel contains the active site residues His270 and Asp306, the C-terminal β-barrel has Ser387 from <scene name='80/801748/Cv/13'>the catalytic triad</scene>. The substantial structural conservation across HtrA enzymes suggests a similar reaction mechanism as evident from the positive charge cavity (the oxyanion hole) which helps in stabilization of tetrahedral intermediate during the acylation step of catalysis. The side chain of the active site serine, Ser387, could be modelled in two alternate conformations with an occupancy of 0.53 and 0.47. Of note, that N<sub>δ1</sub> (His270) and O<sub>δ1</sub>/O<sub>δ2</sub> (Asp306) are within <scene name='80/801748/Cv/14'>hydrogen bonding distance</scene> (2.6Å /3.2Å). On the other hand, the orientation of active site histidine places N<sub>ε2</sub> of His270 <scene name='80/801748/Cv/15'>significantly away</scene> from the O<sub>ϒ</sub> of Ser387. The orientation of H270 (N<sub>ε2</sub>) and Ser387 (O<sub>γ</sub>) (separated by ''ca'' 8.0Å) suggests that this crystal structure represents an inactive conformation. The PDZ domain is linked to the protease domain by a twelve residue long polypeptide segment. Based on extensive analysis of ''E. coli'' DegS, the L1 (157-167) and L3 (141-148) loops are essential for regulation of protease activity whereas the L2 loop governs substrate specificity.<ref name="Hasenbein">PMID:20184896</ref> These loops connecting helices or strands in protease domain are highlighted (green). The movement of L3 loop away from PDZ domain has been shown to shift the equilibrium from the inactive to active state of DegS upon peptide binding to PDZ domain.<ref name="Sohn">PMID:19836340</ref> In the ''M. tuberculosis'' HtrA structure, was noted that the L3 loop is displaced from the PDZ domain. |
<b>References</b><br> | <b>References</b><br> | ||
Revision as of 11:45, 26 November 2018
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