A Key Hydrophobic Patch Identified in an AAA⁺ Protein Essential for Its In Trans Inhibitory Regulation

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• 作者：Nan Zhang ; Timothy Simpson ; Edward Lawton ; Povilas Uzdavinys ; Nicolas Joly ; Patricia Burrows ; Martin Buck
• 关键词：bEBP ; bacterial enhancer binding protein ; AAA+ ; ATPases Associated with various cellular Activities ; PspA ; phage shock protein A ; PspF ; phage shock protein F ; RNAP ; RNA polymerase ; WT ; wild type ; PDB ; Protein Data Bank ; EDTA ; ethylenediaminetetraacetic ac
• 刊名：Journal of Molecular Biology
• 出版年：2013
• 出版时间：9 August, 2013
• 年：2013
• 卷：425
• 期：15
• 页码：2656-2669
• 全文大小：2700 K

文摘

Bacterial enhancer binding proteins (bEBPs) are a subclass of the AAA⁺ (ATPases Associated with various cellular Activities) protein family. They are responsible for ¦Ò⁵⁴-dependent transcription activation during infection and function under many stressful growth conditions. The majority of bEBPs are regulated in their formation of ring-shaped hexameric self-assemblies via an amino-terminal domain through its phosphorylation or ligand binding. In contrast, the Escherichia coli phage shock protein F (PspF) is negatively regulated in trans by phage shock protein A (PspA). Up to six PspA subunits suppress PspF hexamer action. Here, we present biochemical evidence that PspA engages across the side of a PspF hexameric ring. We identify three key binding determinants located in a surface-exposed ¡®W56 loop¡¯ of PspF, which form a tightly packed hydrophobic cluster, the ¡®YLW¡¯ patch. We demonstrate the profound impact of the PspF W56 loop residues on ATP hydrolysis, the ¦Ò⁵⁴ binding loop 1, and the self-association interface. We infer from single-chain studies that for complete PspF inhibition to occur, more than three PspA subunits need to bind a PspF hexamer with at least two binding to adjacent PspF subunits. By structural modelling, we propose that PspA binds to PspF via its first two helical domains. After PspF binding-induced conformational changes, PspA may then share structural similarities with a bEBP regulatory domain.