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

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 GSALTVRDWPALEALAKTMPADAGARAMTDDDLRAAGVDRRVPEQKLGAAIDEFASLRLPDRIDGRFVDGRRANLTVFDDARVAVRGHARAQRNLLERLETELLGGTLDTAGDEGGIQPDPILQGLVDVIGQGKSDIDAYATIVEGLTKYFQSVADVMSKLQDYISAKDDKNMKIDGGKIKALIQQVIDHLPTMQLPKGADIARWRKELGDAVSISDSGVVTINPDKLIKMRDSLPPDGTVWDTARYQAWNTAFSGQKDNIQNDVQTLVEKYSHQNSNFDNLVKVLSGAISTLTDTAKSYLQI ; Mutations for sequence MLNIQNYSASPHPGIVAERPQTPSASEHVETAVVPSTTEHRGTDIISLSQAATKIHQAQQTLQSTPPISEENNDERTLARQQLTSSLNALAKSGVSLSAEQNENLRSAFSAPTSALFSASPMAQPRTTISDAEIWDMVSQNISAIGDSYLGVYENVVAVYTDFYQAFSDILSKMGGWLLPGKDGNTVKLDVTSLKNDLNSLVNKYNQINSNTVLFPAQSGSGVKVATEAEARQWLSELNLPNSCLKSYGSGYVVTVDLTPLQKMVQDIDGLGAPGKDSKLEMDNAKYQAWQSGFKAQEENMKTTLQTLTQKYSNANSLYDNLVKVLSSTISSSLETAKSFLQG ; Mutations for sequence MEVRNFNAARELFLDELLAAPAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLKDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI ; Mutations for sequence GLTGHGSSVLEELVQLVAAANIDISIKYDPRKDSEVFANRVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVMHFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDKNLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEALNRFIQKYDSVMQRLLDDTSGKHHHHHH
Sequence Assay Result Units