Caulobacter S-layer structure and regulation

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We study biogenesis of the protein surface (S)-layer of Caulobacter crescentus. This 2-D crystalline lattice encases the bacterium and is comparable to S-layers of many bacteria and Archaea. This S-layer is one of few exported by type 1 protein secretion. Significance: 1) S-layers. Few are studied in molecular detail and structural principles are poorly understood. 2) Type 1 secretion. An important secretion mechanism for Gram (-) bacteria, especially pathogens, yet few study mechanistic details 3) Biotechnology. Understanding Caulobacter S-layer structure has utility in design and display of heterologous proteins used as human therapeutic agents. The Specific Objectives and Focused Significance are in 4 areas: 1) S-layer structure. Atomic resolution of S-layers is challenging because of difficulties in crystallizing a self-assembling protein. We will dissect the protein into its domains (attachment, crystallization, secretion signal), and by collaboration, crystallize and solve structures. Each region addresses important issues: a) the attachment domain is a specialized lectin and there are few paradigms for surface attachment in Gram (-), b) Type 1 C-terminal secretion signals are poorly understood, with no solved structures and c) the role of the RTX motif of the crystallization domain is not understood for any type 1 secreted protein. 2) Secretion/translation Regulation. Secretion is at extreme levels, but when blocked there is strong posttranscriptional downregulation. We will do site-directed mutagenesis of mRNA translation initiation regions, compare S-layer truncates for expression and examine small RNAs to find the basis of control. 3) Attachment LPS. The S-layer attaches via LPS. We will assess O-side chain composition, an apparently unique arrangement of modified perosamine residues and co-crystallize it with the attachment domain. 4) S-layer associated protease. This protease causes cleavage of S-layer if not properly folded and site-specific cleavage within foreign inserts. We will learn how it attaches to the cell surface and "edits" S-layer.