1bcz: Difference between revisions

Revision as of 12:53, 21 February 2008

RECOMBINANT RAT ANNEXIN V, T72S MUTANT

OverviewOverview

Annexin V belongs to a family of eukaryotic calcium-dependent membrane-binding proteins. The calcium-binding sites at the annexin-membrane interface have been investigated in some detail; however, little is known about the functional roles of highly conserved interfacial residues that do not coordinate calcium themselves. In the present study, the importance of tryptophan 185, and threonine or serine at positions 72, 144, 228, and 303, in rat annexin V is investigated by site-directed mutagenesis, X-ray crystallography, and functional assays. The high-resolution crystal structures of the mutants show that the mutations do not cause structural perturbations of the annexin molecule itself or disappearance of bound calcium ions from calcium-binding sites. The assays indicate that relative to wild-type annexin V, loss of the methyl substituent at position 72 (Thr72-->Ser) has no effect while loss of the hydroxyl group (Thr72-->Ala or Thr72-->Lys) causes reduction of membrane binding. Multiple lysine substitutions (e.g., Thr72,Ser144,Ser228,Ser303-->Lys) have a greater adverse effect than the single lysine mutation, suggesting that in annexin V the introduction of potentially favorable electrostatic interactions between the lysine side chains and the net negatively charged membrane surface is not sufficient to overcome the loss of the hydroxyl side chains. Replacement of the unique tryptophan, Trp185, by alanine similarly decreases membrane binding affinity. Taken together, the data suggest that the side chains mutated in this study contribute to phospholipid binding and participate directly in intermolecular contacts with phospholipid membrane components.

About this StructureAbout this Structure

1BCZ is a Single protein structure of sequence from Rattus norvegicus with and as ligands. Full crystallographic information is available from OCA.

ReferenceReference

Mutational and crystallographic analyses of interfacial residues in annexin V suggest direct interactions with phospholipid membrane components., Campos B, Mo YD, Mealy TR, Li CW, Swairjo MA, Balch C, Head JF, Retzinger G, Dedman JR, Seaton BA, Biochemistry. 1998 Jun 2;37(22):8004-10. PMID:9609693

Page seeded by OCA on Thu Feb 21 11:53:58 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1bcz.jpg|left|200px]]<br /><applet load="1bcz" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1bcz.jpg|left|200px]]<br /><applet load="1bcz" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1bcz, resolution 2.2&Aring;" />
 '''RECOMBINANT RAT ANNEXIN V, T72S MUTANT'''<br />
 ==Overview==
-Annexin V belongs to a family of eukaryotic calcium-dependent, membrane-binding proteins. The calcium-binding sites at the, annexin-membrane interface have been investigated in some detail; however, little is known about the functional roles of highly conserved interfacial, residues that do not coordinate calcium themselves. In the present study, the importance of tryptophan 185, and threonine or serine at positions 72, 144, 228, and 303, in rat annexin V is investigated by site-directed, mutagenesis, X-ray crystallography, and functional assays. The, high-resolution crystal structures of the mutants show that the mutations, do not cause structural perturbations of the annexin molecule itself or, disappearance of bound calcium ions from calcium-binding sites. The assays, indicate that relative to wild-type annexin V, loss of the methyl, substituent at position 72 (Thr72--&gt;Ser) has no effect while loss of the, hydroxyl group (Thr72--&gt;Ala or Thr72--&gt;Lys) causes reduction of membrane, binding. Multiple lysine substitutions (e.g., Thr72,Ser144,Ser228,Ser303--&gt;Lys) have a greater adverse effect than the, single lysine mutation, suggesting that in annexin V the introduction of, potentially favorable electrostatic interactions between the lysine side, chains and the net negatively charged membrane surface is not sufficient, to overcome the loss of the hydroxyl side chains. Replacement of the, unique tryptophan, Trp185, by alanine similarly decreases membrane binding, affinity. Taken together, the data suggest that the side chains mutated in, this study contribute to phospholipid binding and participate directly in, intermolecular contacts with phospholipid membrane components.
+Annexin V belongs to a family of eukaryotic calcium-dependent membrane-binding proteins. The calcium-binding sites at the annexin-membrane interface have been investigated in some detail; however, little is known about the functional roles of highly conserved interfacial residues that do not coordinate calcium themselves. In the present study, the importance of tryptophan 185, and threonine or serine at positions 72, 144, 228, and 303, in rat annexin V is investigated by site-directed mutagenesis, X-ray crystallography, and functional assays. The high-resolution crystal structures of the mutants show that the mutations do not cause structural perturbations of the annexin molecule itself or disappearance of bound calcium ions from calcium-binding sites. The assays indicate that relative to wild-type annexin V, loss of the methyl substituent at position 72 (Thr72--&gt;Ser) has no effect while loss of the hydroxyl group (Thr72--&gt;Ala or Thr72--&gt;Lys) causes reduction of membrane binding. Multiple lysine substitutions (e.g., Thr72,Ser144,Ser228,Ser303--&gt;Lys) have a greater adverse effect than the single lysine mutation, suggesting that in annexin V the introduction of potentially favorable electrostatic interactions between the lysine side chains and the net negatively charged membrane surface is not sufficient to overcome the loss of the hydroxyl side chains. Replacement of the unique tryptophan, Trp185, by alanine similarly decreases membrane binding affinity. Taken together, the data suggest that the side chains mutated in this study contribute to phospholipid binding and participate directly in intermolecular contacts with phospholipid membrane components.
 ==About this Structure==
-BCZ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Rattus_norvegicus Rattus norvegicus] with CA and SO4 as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1BCZ OCA].
+BCZ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Rattus_norvegicus Rattus norvegicus] with <scene name='pdbligand=CA:'>CA</scene> and <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1BCZ OCA].
 ==Reference==
@@ Line 13: / Line 13: @@
 [[Category: Rattus norvegicus]]
 [[Category: Single protein]]
-[[Category: Head, J.F.]]
+[[Category: Head, J F.]]
-[[Category: Li, C.W.]]
+[[Category: Li, C W.]]
-[[Category: Mo, Y.D.]]
+[[Category: Mo, Y D.]]
-[[Category: Seaton, B.A.]]
+[[Category: Seaton, B A.]]
-[[Category: Swairjo, M.A.]]
+[[Category: Swairjo, M A.]]
 [[Category: CA]]
 [[Category: SO4]]
@@ Line 23: / Line 23: @@
 [[Category: phospholipid membrane binding protein]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 11:33:21 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:53:58 2008''