6tyy

From Proteopedia

(Difference between revisions)

Revision as of 15:38, 20 November 2019

Hedgehog autoprocessing mutant D46H

Structural highlights

6tyy is a 1 chain structure. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
NonStd Res:
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

[HH_DROME] Intercellular signal essential for a variety of patterning events during development. Establishes the anterior-posterior axis of the embryonic segments and patterns the larval imaginal disks. Binds to the patched (ptc) receptor, which functions in association with smoothened (smo), to activate the transcription of target genes wingless (wg), decapentaplegic (dpp) and ptc. In the absence of hh, ptc represses the constitutive signaling activity of smo through fused (fu).^[1] ^[2] ^[3] ^[4] The hedgehog protein N-product constitutes the active species in both local and long-range signaling, whereas the C-terminal product has no signaling activity. It acts as a morphogen, and diffuses long distances despite its lipidation. Heparan sulfate proteoglycans of the extracellular matrix play an essential role in diffusion. Lipophorin is required for diffusion, probably by acting as vehicle for its movement, explaining how it can spread over long distances despite its lipidation.^[5] ^[6] ^[7] ^[8] The hedgehog protein C-product, which mediates the autocatalytic activity, has no signaling activity.^[9] ^[10] ^[11] ^[12]

Publication Abstract from PubMed

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 x 10(3) and 3.7 x 10(3) M(-1) s(-1), respectively, and an identical pKa = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (>200-fold) and epicoprostanol (>300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.

General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.,Zhao J, Ciulla DA, Xie J, Wagner AG, Castillo DA, Zwarycz AS, Lin Z, Beadle S, Giner JL, Li Z, Li H, Banavali N, Callahan BP, Wang C J Am Chem Soc. 2019 Nov 7. doi: 10.1021/jacs.9b08914. PMID:31682419^[13]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Tashiro S, Michiue T, Higashijima S, Zenno S, Ishimaru S, Takahashi F, Orihara M, Kojima T, Saigo K. Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication. Gene. 1993 Feb 28;124(2):183-9. PMID:8166882
↑ Lee JJ, von Kessler DP, Parks S, Beachy PA. Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell. 1992 Oct 2;71(1):33-50. PMID:1394430
↑ Tabata T, Eaton S, Kornberg TB. The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 1992 Dec;6(12B):2635-45. PMID:1340474
↑ Lee JD, Kraus P, Gaiano N, Nery S, Kohtz J, Fishell G, Loomis CA, Treisman JE. An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development. Dev Biol. 2001 May 1;233(1):122-36. PMID:11319862 doi:http://dx.doi.org/10.1006/dbio.2001.0218
↑ Tashiro S, Michiue T, Higashijima S, Zenno S, Ishimaru S, Takahashi F, Orihara M, Kojima T, Saigo K. Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication. Gene. 1993 Feb 28;124(2):183-9. PMID:8166882
↑ Lee JJ, von Kessler DP, Parks S, Beachy PA. Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell. 1992 Oct 2;71(1):33-50. PMID:1394430
↑ Tabata T, Eaton S, Kornberg TB. The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 1992 Dec;6(12B):2635-45. PMID:1340474
↑ Lee JD, Kraus P, Gaiano N, Nery S, Kohtz J, Fishell G, Loomis CA, Treisman JE. An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development. Dev Biol. 2001 May 1;233(1):122-36. PMID:11319862 doi:http://dx.doi.org/10.1006/dbio.2001.0218
↑ Tashiro S, Michiue T, Higashijima S, Zenno S, Ishimaru S, Takahashi F, Orihara M, Kojima T, Saigo K. Structure and expression of hedgehog, a Drosophila segment-polarity gene required for cell-cell communication. Gene. 1993 Feb 28;124(2):183-9. PMID:8166882
↑ Lee JJ, von Kessler DP, Parks S, Beachy PA. Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog. Cell. 1992 Oct 2;71(1):33-50. PMID:1394430
↑ Tabata T, Eaton S, Kornberg TB. The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 1992 Dec;6(12B):2635-45. PMID:1340474
↑ Lee JD, Kraus P, Gaiano N, Nery S, Kohtz J, Fishell G, Loomis CA, Treisman JE. An acylatable residue of Hedgehog is differentially required in Drosophila and mouse limb development. Dev Biol. 2001 May 1;233(1):122-36. PMID:11319862 doi:http://dx.doi.org/10.1006/dbio.2001.0218
↑ Zhao J, Ciulla DA, Xie J, Wagner AG, Castillo DA, Zwarycz AS, Lin Z, Beadle S, Giner JL, Li Z, Li H, Banavali N, Callahan BP, Wang C. General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing. J Am Chem Soc. 2019 Nov 7. doi: 10.1021/jacs.9b08914. PMID:31682419 doi:http://dx.doi.org/10.1021/jacs.9b08914

1 Structural highlights
2 Function
3 Publication Abstract from PubMed
4 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/6tyy"

@@ Line 1: / Line 1: @@
-'''Unreleased structure'''
-The entry 6tyy is ON HOLD  until Paper Publication
+==Hedgehog autoprocessing mutant D46H==
+<StructureSection load='6tyy' size='340' side='right'caption='[[6tyy]], [[Resolution|resolution]] 1.36&Aring;' scene=''>
+== Structural highlights ==
+<table><tr><td colspan='2'>[[6tyy]] is a 1 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6TYY OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6TYY FirstGlance]. <br>
+</td></tr><tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=CSD:3-SULFINOALANINE'>CSD</scene></td></tr>
+<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6tyy FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6tyy OCA], [http://pdbe.org/6tyy PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6tyy RCSB], [http://www.ebi.ac.uk/pdbsum/6tyy PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6tyy ProSAT]</span></td></tr>
+</table>
+== Function ==
+[[http://www.uniprot.org/uniprot/HH_DROME HH_DROME]] Intercellular signal essential for a variety of patterning events during development. Establishes the anterior-posterior axis of the embryonic segments and patterns the larval imaginal disks. Binds to the patched (ptc) receptor, which functions in association with smoothened (smo), to activate the transcription of target genes wingless (wg), decapentaplegic (dpp) and ptc. In the absence of hh, ptc represses the constitutive signaling activity of smo through fused (fu).<ref>PMID:8166882</ref> <ref>PMID:1394430</ref> <ref>PMID:1340474</ref> <ref>PMID:11319862</ref>   The hedgehog protein N-product constitutes the active species in both local and long-range signaling, whereas the C-terminal product has no signaling activity. It acts as a morphogen, and diffuses long distances despite its lipidation. Heparan sulfate proteoglycans of the extracellular matrix play an essential role in diffusion. Lipophorin is required for diffusion, probably by acting as vehicle for its movement, explaining how it can spread over long distances despite its lipidation.<ref>PMID:8166882</ref> <ref>PMID:1394430</ref> <ref>PMID:1340474</ref> <ref>PMID:11319862</ref>   The hedgehog protein C-product, which mediates the autocatalytic activity, has no signaling activity.<ref>PMID:8166882</ref> <ref>PMID:1394430</ref> <ref>PMID:1340474</ref> <ref>PMID:11319862</ref>
+<div style="background-color:#fffaf0;">
+== Publication Abstract from PubMed ==
+Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation. Native activity toward cholesterol was established for D46H in vitro using a continuous FRET-based autoprocessing assay and in cellulo with stable expression in human 293T cells. The catalytic efficiency of cholesterylation with D46H is similar to that with wild type (WT), with kmax/KM = 2.1 x 10(3) and 3.7 x 10(3) M(-1) s(-1), respectively, and an identical pKa = 5.8 is obtained for both residues by NMR. To our knowledge this is the first example where a general base substitution of an Asp for His preserves both the structure and activity as a general base. Surprisingly, D46H exhibits increased catalytic efficiency toward non-native substrates, especially coprostanol (&gt;200-fold) and epicoprostanol (&gt;300-fold). Expanded substrate tolerance is likely due to stabilization by H46 of the negatively charged tetrahedral intermediate using electrostatic interactions, which are less constrained by geometry than H-bond stabilization by D46. In addition to providing fundamental insights into Hh autoprocessing, our findings have important implications for protein engineering and enzyme design.
-Authors: Li, H., Li, Z., Wang, C., Callahan, B.P.
+General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.,Zhao J, Ciulla DA, Xie J, Wagner AG, Castillo DA, Zwarycz AS, Lin Z, Beadle S, Giner JL, Li Z, Li H, Banavali N, Callahan BP, Wang C J Am Chem Soc. 2019 Nov 7. doi: 10.1021/jacs.9b08914. PMID:31682419<ref>PMID:31682419</ref>
-Description: Hedgehog autoprocessing mutant D46H
+From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
-[[Category: Unreleased Structures]]
+</div>
+<div class="pdbe-citations 6tyy" style="background-color:#fffaf0;"></div>
+== References ==
+<references/>
+__TOC__
+</StructureSection>
+[[Category: Large Structures]]
+[[Category: Callahan, B P]]
 [[Category: Li, H]]
 [[Category: Li, Z]]
 [[Category: Wang, C]]
-[[Category: Callahan, B.P]]
+[[Category: Hedgehog autoprocessing doamin]]
+[[Category: Signaling protein]]

6tyy

From Proteopedia

Revision as of 15:38, 20 November 2019

Hedgehog autoprocessing mutant D46H

Structural highlights

Function

Publication Abstract from PubMed

References

Contents

Proteopedia Page Contributors and Editors (what is this?)

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