1htj
From Proteopedia
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==Overview== | ==Overview== | ||
| - | BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine | + | BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine nucleotide exchange factors that upregulate Rho GTPases. PDZ-RhoGEF and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to Rho proteins. A approximately 200 residue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with Galpha12/13 proteins. To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human PDZ-RhoGEF. RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine. A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation. A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts. CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions. Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization. |
==About this Structure== | ==About this Structure== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Chikumi, H.]] | [[Category: Chikumi, H.]] | ||
| - | [[Category: Derewenda, Z | + | [[Category: Derewenda, Z S.]] |
| - | [[Category: Gutkind, J | + | [[Category: Gutkind, J S.]] |
| - | [[Category: Lewis, M | + | [[Category: Lewis, M E.]] |
| - | [[Category: Longenecker, K | + | [[Category: Longenecker, K L.]] |
[[Category: gef]] | [[Category: gef]] | ||
[[Category: guanine nucleotide exchange factor]] | [[Category: guanine nucleotide exchange factor]] | ||
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[[Category: rgs-like]] | [[Category: rgs-like]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:04:47 2008'' |
Revision as of 11:04, 21 February 2008
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STRUCTURE OF THE RGS-LIKE DOMAIN FROM PDZ-RHOGEF
Overview
BACKGROUND: The multidomain PDZ-RhoGEF is one of many known guanine nucleotide exchange factors that upregulate Rho GTPases. PDZ-RhoGEF and related family members play a critical role in a molecular signaling pathway from heterotrimeric G protein-coupled receptors to Rho proteins. A approximately 200 residue RGS-like (RGSL) domain in PDZ-RhoGEF and its homologs is responsible for the direct association with Galpha12/13 proteins. To better understand structure-function relationships, we initiated crystallographic studies of the RGSL domain from human PDZ-RhoGEF. RESULTS: A recombinant construct of the RGSL domain was expressed in Escherichia coli and purified, but it did not crystallize. Alternative constructs were designed based on a novel strategy of targeting lysine and glutamic acid residues for mutagenesis to alanine. A triple-point mutant functionally identical to the wild-type protein was crystallized, and its structure was determined by the MAD method using Se-methionine (Se-Met) incorporation. A molecular model of the RGSL domain was refined at 2.2 A resolution, revealing an all-helical tertiary fold with the mutations located at intermolecular lattice contacts. CONCLUSIONS: The first nine helices adopt a fold similar to that observed for RGS proteins, although the sequence identity with other such known structures is below 20%. The last three helices are an integral extension of the RGS fold, packing tightly against helices 3 and 4 with multiple hydrophobic interactions. Comparison with RGS proteins suggests features that are likely relevant for interaction with G proteins. Finally, we conclude that the strategy used to produce crystals was beneficial and might be applicable to other proteins resistant to crystallization.
About this Structure
1HTJ is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
Reference
Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric g protein-coupled signaling to Rho GTPases., Longenecker KL, Lewis ME, Chikumi H, Gutkind JS, Derewenda ZS, Structure. 2001 Jul 3;9(7):559-69. PMID:11470431
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