Structure-based mutagenesis of SigE verifies the importance of hydrophobic and electrostatic residues in type III chaperone function.
Despite sharing little sequence identity, most type III chaperones display a similar homodimeric structure characterized by negative charges distributed broadly over their entire surface, interspersed with hydrophobic patches. Here we have used SigE from Salmonella as a model for class IA type III chaperones to investigate the role of these surface-exposed residues in chaperone function. SigE is essential for the stability, secretion and translocation of its cognate effector, SopB (SigD). We analysed the effect of mutating nine conserved hydrophobic and electronegative surface-exposed amino acids of SigE on SopB binding, stability, secretion and translocation. Six of these mutations affected some aspect of SigE function (Leu14, Asp20, Leu22, Leu23, Ile25 and Asp51) and three were without effect (Leu54, Glu92 and Glu99). Our results highlight that both hydrophobic and electronegative surfaces are required for the function of SigE and provide an important basis for the prediction of side-chain requirements for other chaperone-effector pairs.
Knodler
Leigh A
LA
Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA. lknodler@niaid.nih.gov
Bertero
Michela
M
Yip
Calvin
C
Strynadka
Natalie C J
NC
Steele-Mortimer
Olivia
O
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003461:Crystallography; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000737:chemistry / Q000378:metabolism*; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D012808:Sigma Factor; C111259:sigE protein, Bacteria; C489224:SopB protein, Salmonella