User:Fadel A. Samatey/FlhBc I: Difference between revisions

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[[Interactive_3D_Complement_in_Proteopedia|Interactive 3D Complement in Proteopedia]]

[[Interactive_3D_Complement_in_Proteopedia|Interactive 3D Complement in Proteopedia]]

[[Image:Acta_Cryst_D.png|900 px]]

Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins.

Vladimir A. Meshcheryakov, Akio Kitao, Hideyuki Matsunami and Fadel A. Samatey. ''Acta Cryst. D69: 812-820 (2013). [http://dx.doi.org/10.1107/S0907444913002102 doi:10.1107/S0907444913002102]

Vladimir A. Meshcheryakov, Akio Kitao, Hideyuki Matsunami and [[Fadel A. Samatey Group|Fadel A. Samatey]] ([[Fadel A. Samatey Group (Japanese)|サマテ]]). ''Acta Cryst. D69: 812-820 (2013). [http://dx.doi.org/10.1107/S0907444913002102 doi:10.1107/S0907444913002102] Open Access.

Contact: [[Image:Samatey-email-address.png|160px]]

===Brief Introduction===

FlhB is a membrane protein that is part of the flagellum-specific secretion apparatus. It is required for secretion of flagellar proteins, and for bacterial motility. FlhB is paralogous to a protein in the virulence type III secretion system. FlhB has a hydrophobic integral membrane domain, predicted to have four transmembrane helices, a flexible linker that is highly conserved and essential for function, and a cytoplasmic domain. The present study reports the structures of the cytoplasmic domains of two bacterial taxa.

(Please see [http://dx.doi.org/10.1107/S0907444913002102 the publication] for a more detailed introduction.)

(Please see [http://dx.doi.org/10.1107/S0907444913002102 the open access publication] for a more detailed introduction.)

==Molecular Tour: FlhBc Structures==

<!--<table cellpadding="6" style="background-color:#ffff80"><tr><td>

This page is under construction. We expect to complete additional interactive molecular scenes before May 5, 2013.

</td></tr></table>

</td></tr></table>-->

====''Salmonella''====

===''Salmonella''===

FlhB from ''Salmonella typhimurium'' consists of 383 amino acids. The cytoplasmic domain 219-383 (length 165, 43% of full length) was crystallized. The resulting model [[3b0z]] includes coordinates for residues 229-353 (length 125, 76% of the crystallized length). The [[asymmetric unit]] contains a single molecule (<scene name='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st/6'>restore initial scene</scene>). As explained in the publication, the position of the long alpha helix appears to be stabilized by

FlhB from ''Salmonella typhimurium'' consists of 383 amino acids. The cytoplasmic domain 219-383 (length 165, 43% of full length) was crystallized. The resulting model [[3b0z]] includes coordinates for residues 229-353 (length 125, 76% of the crystallized length). The [[asymmetric unit]] contains a single molecule (<scene name='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st/6'>restore initial scene</scene>).

<center>{{Template:ColorKey_Helix}},

{{Template:ColorKey_Strand}}</center>

As explained in the publication, the position of the long alpha helix appears to be stabilized by

[[crystal contacts]] (not shown).

The chain is <scene name='User:Fadel_A._Samatey/FlhBc_I/Cleavage/1'>cleaved at NPTH between Asn269 and Pro270</scene>. This is believed to be autocatalytic cleavage involved in the transition of the export apparatus from hook to filament mode. Mutations that prevent this cleavage render the bacteria non-motile.

====''Aquifex''====

===''Aquifex''===

FlhB from the thermophile<ref>[http://en.wikipedia.org/wiki/Aquifex_aeolicus Optimum growth ~90o C].</ref> ''Aquifex aeolicus'' is shorter, 350 residues (vs. 383 for ''S. typhimurium''), with 32% sequence identity. Residues 213-350 (length 138) were crystallized, and the resulting model [[3b1s]] has <scene name='User:Fadel_A._Samatey/FlhBc_I/Triple-au-aquifex/1'>three molecules</scene> in the [[asymmetric unit]]. The molecule displayed in the comparison in the next section, with chains designated C and D, was chosen because it has the lowest average [[temperature factor]] (66.2, vs. 84.7 and 72.6 for A,B and E,F respectively). It has coordinates for 232-337 (length 106, 77% of the crystallized segment), cleaved at NPTH between Asn263 and Pro264.

====Comparison====

===Comparison===

The FlhBc ''Salmonella'' 3D structure is very similar to that of ''Aquifex''. 102 alpha carbons align with an RMSD of 1.0 Å. Their FlhB's have 32% sequence identity.

<scene name='User:Fadel_A._Samatey/FlhBc_I/Flhbc_st_plus_aa/2'>Display both structures</scene>, then click the button below to do a structural alignment.

<jmol>

<text>Do Structural Alignment</text>

</jmolButton>

</jmol>

<center>Salmonella vs. Aquifex.</center>

===Charge===

The <scene name='User:Fadel_A._Samatey/FlhBc_I/Charge_st/2'>charges on the surface of FlhBc</scene> (''S. typhimurium'') show a cluster of positive charges on one side, referred to in the publication as a "positively charged cleft".

<center>{{Template:ColorKey_Charge_Anionic}} / {{Template:ColorKey_Charge_Cationic}} / '''Histidine'''</center>

However, only two of the positively charged residues in this cleft region are conserved: Arg320 (ConSurf level 9, maximum), and Arg245 (Consurf level 8). This is shown in another scene below under ''Evolutionary Conservation''.

===Loop 281-285===

Both FlhBc structures have a <scene name='User:Fadel_A._Samatey/FlhBc_I/281-285/3'>protruding loop, which in ''Salmonella'' is 281-285, ENKMS</scene>. Despite the fact that this loop is '''not conserved''' (see below), its deletion abolished motility. Mutation of the loop to PPPPP reduced motility, while mutation to AAAAA had no effect. The reductions in motility correlated with reductions in secretion of hook protein FlgE and filament protein FliC.

===Flexibility of N-Terminus===

Molecular dynamics simulations suggested that the mutations in 281-285 reduced flexibility of the N-terminal alpha helix, and hence that such flexibility may be important for function. Indeed, 226-267 (the N-terminal helix is 229 to about 262) is predicted to be intrinsically disordered<ref>Prediction of intrinsic disorder for ''Salmonella typhimurium'' FlhB by the FoldIndex server (image below, at right). [[Image:3b0z-FlhB-foldindex.png|frame]]</ref>. The observed formation of a helix seems likely to depend upon stabilization by crystal contacts mentioned above.

===Evolutionary Conservation===

<scene name='User:Fadel_A._Samatey/FlhBc_I/Conservation_st/1'>Conservation of FlhBc (''S. typhimurium'')</scene> was calculated by the [http://consurf.tau.ac.il ConSurf Server].

<center>{{Template:ColorKey_ConSurf_NoYellow_NoGray}}</center>

There is a prominent strip of conserved residues on one side of the long N-terminal alpha helix. As mentioned above, the auto-catalytically cleaved segment, including 269 and 270, is highly conserved. In contrast (as mentioned above) the protruding loop 281-285 (which is necessary for function and suggested to be important for flexibility of the N terminus) is not conserved.

Conservation of ''A. aeolicus'' FlhBc (not shown) was similar, especially in the regions mentioned above.

Turning to the positive charges discussed above, <scene name='User:Fadel_A._Samatey/FlhBc_I/Conservation_st/3'>here only positively charged sidechains (Lys, Arg) are spacefilling. His is depicted in sticks.</scene> All atoms are colored by conservation. With spinning off, touch any sidechain to report its identity.

</StructureSection>

===References and Notes===

* If you have any comments, questions, or if there is any problem with this page, please contact [[Image:Samatey-email-address.png|150px]].

<!--

===Notes for Developers===

*[[Image:Workbench 3b0z.pdb.gz]]

*[[Image:Workbench 3b1s.pdb.gz]]

*[[Image:Workbench 3b0z 3b1sCD.pdb]] 1.1 is 3b0z(St) while 1.2 is 3b1s chains C and D. Not aligned.

*[[Image:Workbench 3b0z 3b1sCD aligned.pdb]]

*[[Image:Workbench 3b0z 3b1sCD aligned.pdb]] -->

__NOTOC__

@@ Line 1: / Line 1: @@
-[[Interactive_3D_Complement_in_Proteopedia|Interactive 3D Complement in Proteopedia]]
+[[Interactive_3D_Complement_in_Proteopedia|Interactive 3D Complement in Proteopedia]]<br>
 [[Image:Acta_Cryst_D.png|900 px]]
 <br><br>
 <span style="font-size:160%"><b>Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins.</b></span><br><br>
-<span style="font-size:120%">Vladimir A. Meshcheryakov, Akio Kitao, Hideyuki Matsunami and Fadel A. Samatey. ''Acta Cryst. D69: 812-820 (2013). [http://dx.doi.org/10.1107/S0907444913002102 doi:10.1107/S0907444913002102]</span>
+<span style="font-size:120%">Vladimir A. Meshcheryakov, Akio Kitao, Hideyuki Matsunami and [[Fadel A. Samatey Group|Fadel A. Samatey]] ([[Fadel A. Samatey Group (Japanese)|サマテ]]). ''Acta Cryst. D69: 812-820 (2013). [http://dx.doi.org/10.1107/S0907444913002102 doi:10.1107/S0907444913002102] <span style="background-color:#ffff80;padding:3px;"><i>Open Access.</i></span></span>
+<br>
+Contact: [[Image:Samatey-email-address.png|160px]]
 ===Brief Introduction===
 FlhB is a membrane protein that is part of the flagellum-specific secretion apparatus. It is required for secretion of flagellar proteins, and for bacterial motility. FlhB is paralogous to a protein in the virulence type III secretion system. FlhB has a hydrophobic integral membrane domain, predicted to have four transmembrane helices, a flexible linker that is highly conserved and essential for function, and a cytoplasmic domain. The present study reports the structures of the cytoplasmic domains of two bacterial taxa.
-(Please see [http://dx.doi.org/10.1107/S0907444913002102 the publication] for a more detailed introduction.)
+(Please see [http://dx.doi.org/10.1107/S0907444913002102 the open access publication] for a more detailed introduction.)
 ==Molecular Tour: FlhBc Structures==
-<StructureSection size='450' frame='true' align='right' caption='Cytoplasmic domain of FlhB' scene='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st/6' >
+<!--<StructureSection size='400' frame='true' align='right' caption='Cytoplasmic domain of FlhB' scene='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st/6' >-->
-<table cellpadding="6" style="background-color:#ffff80"><tr><td>
+<StructureSection load='' size='400' side='right' caption='Cytoplasmic domain of FlhB' scene='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st/6'>
+<!--<table cellpadding="6" style="background-color:#ffff80"><tr><td>
 This page is under construction. We expect to complete additional interactive molecular scenes before May 5, 2013.
-</td></tr></table>
+</td></tr></table>-->
-====''Salmonella''====
+===''Salmonella''===
-FlhB from ''Salmonella typhimurium'' consists of 383 amino acids. The cytoplasmic domain 219-383 (length 165, 43% of full length) was crystallized. The resulting model [[3b0z]] includes coordinates for residues 229-353 (length 125, 76% of the crystallized length). The [[asymmetric unit]] contains a single molecule (<scene name='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st/6'>restore initial scene</scene>). As explained in the publication, the position of the long alpha helix appears to be stabilized by
+FlhB from ''Salmonella typhimurium'' consists of 383 amino acids. The cytoplasmic domain 219-383 (length 165, 43% of full length) was crystallized. The resulting model [[3b0z]] includes coordinates for residues 229-353 (length 125, 76% of the crystallized length). The [[asymmetric unit]] contains a single molecule (<scene name='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st/6'>restore initial scene</scene>).
+<center><span style="background-color:black;padding:4px;">{{Template:ColorKey_Helix}},
+{{Template:ColorKey_Strand}}</span></center>
+As explained in the publication, the position of the long alpha helix appears to be stabilized by
 [[crystal contacts]] (not shown).
 <!--<scene name='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st_xtl_contacts/2'>crystal contacts</scene> (use the ''popup'' button to see details).-->
 The chain is <scene name='User:Fadel_A._Samatey/FlhBc_I/Cleavage/1'>cleaved at NPTH between Asn269 and Pro270</scene>. This is believed to be autocatalytic cleavage involved in the transition of the export apparatus from hook to filament mode. Mutations that prevent this cleavage render the bacteria non-motile.
+{{Template:ColorKey_N2CRainbow}}
-====''Aquifex''====
+===''Aquifex''===
 FlhB from the thermophile<ref>[http://en.wikipedia.org/wiki/Aquifex_aeolicus Optimum growth ~90<sup>o</sup> C].</ref> ''Aquifex aeolicus'' is shorter, 350 residues (vs. 383 for ''S. typhimurium''), with 32% sequence identity. Residues 213-350 (length 138) were crystallized, and the resulting model [[3b1s]] has <scene name='User:Fadel_A._Samatey/FlhBc_I/Triple-au-aquifex/1'>three molecules</scene> in the [[asymmetric unit]]. The molecule displayed in the comparison in the next section, with chains designated C and D, was chosen because it has the lowest average [[temperature factor]] (66.2, vs. 84.7 and 72.6 for A,B and E,F respectively). It has coordinates for 232-337 (length 106, 77% of the crystallized segment), cleaved at NPTH between Asn263 and Pro264.
-====Comparison====
+===Comparison===
 The FlhBc ''Salmonella'' <!--<scene name='User:Fadel_A._Samatey/Workbench/I3DC-1/Flhb_st_aa_aligned/2'>-->3D structure is very similar to that of ''Aquifex''. 102 alpha carbons align with an RMSD of 1.0 &Aring;. Their FlhB's have 32% sequence identity.
 <scene name='User:Fadel_A._Samatey/FlhBc_I/Flhbc_st_plus_aa/2'>Display both structures</scene>, then click the button below to do a structural alignment.
 <jmol>
 <jmolButton>
-<script>if ({1.2}.count > 0); compare {1.1} {1.2} SUBSET {*.ca} {246-269:A} {240-263:C} {272-280:B} {266-274:D} {285-351:B} {279-345:D} ATOMS rotate translate; zoomto *1.5;spin y; else; set echo bottom left; color echo white; font echo 20 sansserif;echo "Please display both structures first!"; delay 4; set echo off; endif;</script>
+<script>if ({1.2}.count > 0); compare {1.1} {1.2} SUBSET {*.ca} {246-269:A} {240-263:C} {272-280:B} {266-274:D} {285-351:B} {279-345:D} ATOMS rotate translate 3; zoomto *1.5;spin y; else; set echo bottom left; color echo white; font echo 20 sansserif;echo "Please display both structures first!"; delay 4; set echo off; endif;</script>
 <text>Do Structural Alignment</text>
 </jmolButton>
 </jmol>
+<center><span style="background-color:black;color:white;padding:4px;"><b><font color="#3030ff">S</font><font color="#30a0ff">al</font><font color="#00ff80">m</font><font color="#00ff00">on</font><font color="#e0ff00">e</font><font color="#ff8000">ll</font><font color="#ff0000">a</font> <font color="#909090">vs.</font> Aquifex</b>.</span></center>
+===Charge===
+The <scene name='User:Fadel_A._Samatey/FlhBc_I/Charge_st/2'>charges on the surface of FlhBc</scene> (''S. typhimurium'') show a cluster of positive charges on one side, referred to in the publication as a &quot;positively charged cleft&quot;.
+<center>{{Template:ColorKey_Charge_Anionic}} / {{Template:ColorKey_Charge_Cationic}} / <font color='#8080d0'>'''Histidine'''</font></center>
+However, only two of the positively charged residues in this cleft region are conserved: Arg320 (ConSurf level 9, maximum), and Arg245 (Consurf level 8). This is shown in another scene below under ''Evolutionary Conservation''.
+===Loop 281-285===
+Both FlhBc structures have a <scene name='User:Fadel_A._Samatey/FlhBc_I/281-285/3'>protruding loop, which in ''Salmonella'' is 281-285, ENKMS</scene>. Despite the fact that this loop is '''not conserved''' (see below), its deletion abolished motility. Mutation of the loop to PPPPP reduced motility, while mutation to AAAAA had no effect. The reductions in motility correlated with reductions in secretion of hook protein FlgE and filament protein FliC.
+===Flexibility of N-Terminus===
+Molecular dynamics simulations suggested that the mutations in 281-285 reduced flexibility of the N-terminal alpha helix, and hence that such flexibility may be important for function. Indeed, 226-267 (the N-terminal helix is 229 to about 262) is predicted to be intrinsically disordered<ref>Prediction of intrinsic disorder for ''Salmonella typhimurium'' FlhB by the FoldIndex server (image below, at right). [[Image:3b0z-FlhB-foldindex.png|frame]]</ref>. The observed formation of a helix seems likely to depend upon stabilization by crystal contacts mentioned above.
+===Evolutionary Conservation===
+<scene name='User:Fadel_A._Samatey/FlhBc_I/Conservation_st/1'>Conservation of FlhBc (''S. typhimurium'')</scene> was calculated by the [http://consurf.tau.ac.il ConSurf Server].
+<center>{{Template:ColorKey_ConSurf_NoYellow_NoGray}}</center>
+There is a prominent strip of conserved residues on one side of the long N-terminal alpha helix. As mentioned above, the auto-catalytically cleaved segment, including 269 and 270, is highly conserved. In contrast (as mentioned above) the protruding loop 281-285 (which is necessary for function and suggested to be important for flexibility of the N terminus) is not conserved.
+Conservation of ''A. aeolicus'' FlhBc (not shown) was similar, especially in the regions mentioned above.
+Turning to the positive charges discussed above, <scene name='User:Fadel_A._Samatey/FlhBc_I/Conservation_st/3'>here only positively charged sidechains (Lys, Arg) are spacefilling. His is depicted in sticks.</scene> All atoms are colored by conservation. With spinning off, touch any sidechain to report its identity.
 </StructureSection>
 ===References and Notes===
+* If you have any comments, questions, or if there is any problem with this page, please contact [[Image:Samatey-email-address.png|150px]].
 <references />
+<!--
 ===Notes for Developers===
 *[[Image:Workbench 3b0z.pdb.gz]]
 *[[Image:Workbench 3b1s.pdb.gz]]
 *[[Image:Workbench 3b0z 3b1sCD.pdb]] 1.1 is 3b0z(St) while 1.2 is 3b1s chains C and D. Not aligned.
-*[[Image:Workbench 3b0z 3b1sCD aligned.pdb]]
+*[[Image:Workbench 3b0z 3b1sCD aligned.pdb]] -->
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