The Protein Journal

, Volume 31, Issue 6, pp 487–498 | Cite as

YspC: A Unique Translocator Exhibits Structural Alteration in the Complex form with Chaperone SycB

Article

Abstract

YspC is an annotated translocator of Yersinia secretion apparatus-Yersinia secretion protein type three secretion system of Yersinia enterocolitica, it forms an 1:1 complex with its cognate chaperone SycB. Unlike other translocators, YspC is highly soluble inspite of having a transmembrane region. Size exclusion chromatography shows that YspC exists predominantly in a monomeric form. Multiple sequence alignment and ConSurf (a web based bioinformatic tool) analysis confirm its significant deviation from the closest class of minor translocators. YspC also possesses a tertiary structure signal seen from near UV CD, further confirming its unique nature amongst the groups of translocators. Far UV CD depicts that YspC is predominantly an α-helical protein; however, its secondary structure alters in the YspC-SycB complex. Thermal denaturation curve predicts a cooperative melting behaviour for YspC which is altered in the YspC-SycB complex. Furthermore, trypsinolysis data confirms a different digestion pattern for YspC in isolation, when compared to the complex form with SycB. From the Forsters resonance energy transfer analysis, it can be predicted that the two tetratricopeptide repeat regions of SycB are masked while it forms a complex with YspC and this is further confirmed by the interaction studies of YspC with two truncated forms of SycB. YspC interacted with ∆SycB(1–114) and ∆SycB(36–114) (possessing only the two TPR regions). However, the complexes formed between YspC and truncated forms of SycB have altered physiological states.

Keywords

Ysa-Ysp type three secretion system Minor translocator Far and near UV CD Tyrosine fluorescence and FRET TPRs of SycB 

Abbreviations

TTSS

Type three secretion system

Ysa-Ysp

Yersinia secretion apparatus-Yersinia secretion protein

TPR

Tetratricopeptide repeat

CD

Circular dichroism

ANS

8-Anilinonapthalene-1-sulfonate

FRET

Forsters resonance energy transfer

SEC

Size exclusion chromatography

MSA

Multiple sequence alignment

TB

Tartoff-Hobbs broth

IPTG

Isopropyl β-D-1-thiogalactopyranoside

Ni–NTA

Nickel–nitrilotriacetic acid

BLAST

Basic local alignment search tool

Notes

Acknowledgments

The research was funded by grants from the Department of Science and Technology, Government of India. Indian Institute of Chemical Biology (IICB), a unit of Council of Scientific & Industrial Research provided the facilities and fellowships for this research.

Supplementary material

10930_2012_9426_MOESM1_ESM.docx (739 kb)
Multiple sequence alignment of YspC and other five minor translocators like IpaC, SipC, AopD, PopD and YopD, edited in ESPript. SipC and IpaC were aligned individually and with YspC. Similarly, AopD, PopD and YopD were aligned individually and with YspC (DOCX 738 kb)
10930_2012_9426_MOESM2_ESM.tif (8 mb)
Mass spectrum of YspC digested by trypsin, using MALDI-TOF. YspC was digested by trypsin for 2 h and subjected to native mass spectrometry. Within a range two peaks were detected corresponding to digested two fragments with masses 14550.4297 Da and 12638.7012 Da, respectively (TIFF 8231 kb)

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Abhishek Basu
    • 1
  • Rakesh Chatterjee
    • 1
  • Saumen Datta
    • 1
  1. 1.Structural Biology and Bioinformatics DivisionIndian Institute of Chemical BiologyKolkataIndia

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