Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems.


Abstract

Diverse Gram-negative bacteria use type III secretion systems (T3SS) to translocate effector proteins into the cytoplasm of eukaryotic cells. The type III secretion apparatus (T3SA) consists of a basal body spanning both bacterial membranes and an external needle. A sensor protein lies at the needle tip to detect environmental signals that trigger type III secretion. The Shigella flexneri T3SA needle tip protein, invasion plasmid antigen D (IpaD), possesses two independently folding domains in vitro. In this study, the solution behavior and thermal unfolding properties of IpaD's functional homologs SipD (Salmonella spp.), BipD (Burkholderia pseudomallei), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa) were examined to identify common features within this protein family. CD and FTIR data indicate that all members within this group are alpha-helical with properties consistent with an intramolecular coiled-coil. SipD showed the most complex unfolding profile consisting of two thermal transitions, suggesting the presence of two independently folding domains. No evidence of multiple folding domains was seen, however, for BipD, LcrV, or PcrV. Thermal studies, including DSC, revealed significant destabilization of LcrV, PcrV, and BipD after N-terminal deletions. This contrasted with SipD and IpaD, which behaved like two-domain proteins. The results suggest that needle tip proteins share significant core structural similarity and thermal stability that may be the basis for their common function. Moreover, IpaD and SipD possess properties that distinguish them from the other tip proteins. Study holds ProTherm entries: 22404, 22405, 22406, 22407, 22408, 22409, 22410, 22411, 22412, 22413, 22414, 22415, 22416, 22417, 22418, 22419, 22420, 22421, 22422, 22423, 22424, 22425, 22426, 22427, 22428, 22429, 22430, 22431, 22432, 22433, 22434, 22435, 22436, 22437, 22438, 22439, 22440, 22441, 22442, 22443, 22444, 22445, 22446, 22447, 22448, 22449, 22450, 22451, 22452, 22453, 22454, 22455, 22456, 22457, 22458, 22459, 22460, 22461 Extra Details: Transition 1 tip proteins; T3SS; biophysical analysis; IpaD; SipD; BipD; LcrV; PcrV

Submission Details

ID: GS98ch8t

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:53 p.m.

Version: 1

Publication Details
Espina M;Ausar SF;Middaugh CR;Baxter MA;Picking WD;Picking WL,Protein Sci. (2007) Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems. PMID:17327391
Additional Information

Study Summary

Number of data points 58
Proteins Virulence-associated V antigen ; Virulence-associated V antigen ; Type III secretion protein PcrV ; Cell invasion protein SipD ; Translocator protein BipD ; Translocator protein BipD
Unique complexes 4
Assays/Quantities/Protocols Experimental Assay: Tm prot_conc:2 mg/mL, ionic:NaCl: 150 mM, buffers:Na2HPO4: 10 mM, pH:7.0 ; Experimental Assay: Tm pH:7.4, ionic:: , buffers:Phosphate: 20 mM, prot_conc:0.1 mg/mL ; Experimental Assay: Tm buffers:Phosphate: 10 mM, pH:7.4, ionic:: , prot_conc:0.1 mg/mL ; Experimental Assay: Tm buffers:Phosphate: 10 mM, pH:7.4, ionic:: , prot_conc:0.1 mg/mL ; Experimental Assay: Tm buffers:Phosphate: 10 mM, pH:7.4, ionic:: , prot_conc:0.1 mg/mL
Libraries Mutations for sequence GLTGHGSSVLEELVQLVAAANIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGKHHHHHH ; Mutations for sequence MEVRNFNAARELFLDELLAAPAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLKDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI ; Mutations for sequence MLNIQNYSASPHPGIVAERPQTPSASEHVETAVVPSTTEHRGTDIISLSQAATKIHQAQQTLQSTPPISEENNDERTLARQQLTSSLNALAKSGVSLSAEQNENLRSAFSAPTSALFSASPMAQPRTTISDAEIWDMVSQNISAIGDSYLGVYENVVAVYTDFYQAFSDILSKMGGWLLPGKDGNTVKLDVTSLKNDLNSLVNKYNQINSNTVLFPAQSGSGVKVATEAEARQWLSELNLPNSCLKSYGSGYVVTVDLTPLQKMVQDIDGLGAPGKDSKLEMDNAKYQAWQSGFKAQEENMKTTLQTLTQKYSNANSLYDNLVKVLSSTISSSLETAKSFLQG ; Mutations for sequence GSALTVRDWPALEALAKTMPADAGARAMTDDDLRAAGVDRRVPEQKLGAAIDEFASLRLPDRIDGRFVDGRRANLTVFDDARVAVRGHARAQRNLLERLETELLGGTLDTAGDEGGIQPDPILQGLVDVIGQGKSDIDAYATIVEGLTKYFQSVADVMSKLQDYISAKDDKNMKIDGGKIKALIQQVIDHLPTMQLPKGADIARWRKELGDAVSISDSGVVTINPDKLIKMRDSLPPDGTVWDTARYQAWNTAFSGQKDNIQNDVQTLVEKYSHQNSNFDNLVKVLSGAISTLTDTAKSYLQI

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3NFT 2010-07-14 1.51 Near-atomic resolution analysis of BipD- A component of the type-III secretion system of Burkholderia pseudomallei
3NZZ 2010-11-17 1.65 Crystal Structure of the Salmonella Type III Secretion System Tip Protein SipD
4JBU 2013-05-08 1.65 1.65A structure of the T3SS tip protein LcrV (G28-D322, C273S) from Yersinia pestis
3O00 2010-11-17 1.85 Crystal Structure of the Salmonella Type III Secretion System Tip Protein SipD-C244S
3O01 2010-11-17 1.9 The Crystal Structure of the Salmonella Type III Secretion System Tip Protein SipD in Complex with Deoxycholate
3O02 2010-11-17 1.9 The Crystal Structure of the Salmonella Type III Secretion System Tip Protein SipD in Complex with Chenodeoxycholate
2IZP 2006-09-06 2.1 BipD - an invasion prtein associated with the type-III secretion system of Burkholderia pseudomallei.
1R6F 2004-03-09 2.17 The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague
3ZQE 2011-08-17 2.19 PrgI-SipD from Salmonella typhimurium in complex with deoxycholate
3ZQB 2011-08-17 2.4 PrgI-SipD from Salmonella typhimurium
2IXR 2006-11-02 2.6 BipD of Burkholderia Pseudomallei
2J9T 2006-11-20 2.7 BipD of Burkholderia Pseudomallei
2YM9 2011-08-17 3.0 SipD from Salmonella typhimurium
2YM0 2011-08-17 3.0 Truncated SipD from Salmonella typhimurium

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.9 Virulence-associated V antigen P23994 LCRV_YERPS
99.1 Virulence-associated V antigen A9R9H4 LCRV_YERPG
100.0 Virulence-associated V antigen A4TSQ1 LCRV_YERPP
100.0 Virulence-associated V antigen P0C7U7 LCRV_YERPE
100.0 Cell invasion protein SipD Q56026 SIPD_SALTY
99.7 Translocator protein BipD A3P6Z4 BIPD_BURP0
100.0 Translocator protein BipD Q63K37 BIPD_BURPS
100.0 Translocator protein BipD A3NLD2 BIPD_BURP6
100.0 Translocator protein BipD Q3JL26 BIPD_BURP1
100.0 Translocator protein BipD Q62B10 BIPD_BURMA
100.0 Translocator protein BipD A2S1Q3 BIPD_BURM9
100.0 Translocator protein BipD A3MCH1 BIPD_BURM7