1nub
From Proteopedia
(New page: 200px<br /> <applet load="1nub" size="450" color="white" frame="true" align="right" spinBox="true" caption="1nub, resolution 2.80Å" /> '''HELIX C DELETION MU...) |
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| - | [[Image:1nub.gif|left|200px]]<br /> | + | [[Image:1nub.gif|left|200px]]<br /><applet load="1nub" size="350" color="white" frame="true" align="right" spinBox="true" |
| - | <applet load="1nub" size=" | + | |
caption="1nub, resolution 2.80Å" /> | caption="1nub, resolution 2.80Å" /> | ||
'''HELIX C DELETION MUTANT OF BM-40 FS-EC DOMAIN PAIR'''<br /> | '''HELIX C DELETION MUTANT OF BM-40 FS-EC DOMAIN PAIR'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The extracellular calcium-binding domain (positions 138-286) of the matrix | + | The extracellular calcium-binding domain (positions 138-286) of the matrix protein BM-40 possesses a binding epitope of moderate affinity for several collagen types. This epitope was predicted to reside in helix alphaA and to be partially masked by helix alphaC. Here we show that deletion of helix alphaC produces a 10-fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix. The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 A resolution. This constitutively activated mutant was used to map the collagen-binding site following alanine mutagenesis at 13 positions. Five residues were crucial for binding, R149 and N156 in helix alphaA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM-40. These residues are spatially close and form a flat ring of 15 A diameter which matches the diameter of a triple-helical collagen domain. The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens. Selected mutations in the non-activated mutant DeltaI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM-40. |
==About this Structure== | ==About this Structure== | ||
| - | 1NUB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with CA as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http:// | + | 1NUB is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=CA:'>CA</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1NUB OCA]. |
==Reference== | ==Reference== | ||
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[[Category: site-directed mutagenesis]] | [[Category: site-directed mutagenesis]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:10:05 2008'' |
Revision as of 12:10, 21 February 2008
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HELIX C DELETION MUTANT OF BM-40 FS-EC DOMAIN PAIR
Overview
The extracellular calcium-binding domain (positions 138-286) of the matrix protein BM-40 possesses a binding epitope of moderate affinity for several collagen types. This epitope was predicted to reside in helix alphaA and to be partially masked by helix alphaC. Here we show that deletion of helix alphaC produces a 10-fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix. The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 A resolution. This constitutively activated mutant was used to map the collagen-binding site following alanine mutagenesis at 13 positions. Five residues were crucial for binding, R149 and N156 in helix alphaA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM-40. These residues are spatially close and form a flat ring of 15 A diameter which matches the diameter of a triple-helical collagen domain. The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens. Selected mutations in the non-activated mutant DeltaI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM-40.
About this Structure
1NUB is a Single protein structure of sequence from Homo sapiens with as ligand. Full crystallographic information is available from OCA.
Reference
Crystal structure and mapping by site-directed mutagenesis of the collagen-binding epitope of an activated form of BM-40/SPARC/osteonectin., Sasaki T, Hohenester E, Gohring W, Timpl R, EMBO J. 1998 Mar 16;17(6):1625-34. PMID:9501084
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