Staphylococcal biofilm-forming protein has a contiguous rod-like structure

Abstract

Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein ( S. epidermidis ) promote biofilm formation through their “B” regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn 2+ -mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E–G5 and G5–E–G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn 2+ does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the “zinc zipper.” Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.

Disorder drives cooperative folding in a multidomain protein

“…1). We have previously shown that the E domain is fully unfolded in isolation (10). Because SasG domains have no tryptophans, (un)folding was followed by monitoring intrinsic tyrosine fluorescence.…”

IsdC from Staphylococcus lugdunensis Induces Biofilm Formation under Low-Iron Growth Conditions

“…The length of the B region was shown to be critical for biofilm development because only SasG (or Aap) constructs with five or more B-repeats supported biofilm formation, while constructs with four or fewer B-repeats did not. Structural studies revealed that Aap B-repeats of the appropriate length adopt an elongated, rope-like structure coordinated by zinc ions and wrap around one another in an antiparallel fashion to form bundles of fibers that establish homophilic interactions and have the potential to interconnect neighbor cells (46,47).…”

“…The structural characterization of Aap and SasG in biofilm development has been recently defined (46,47). Aap and SasG comprise an N-terminal A region and repeated B domains toward the C terminus.…”

Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

“…1). Several studies have demonstrated that Aap is a rod-like fibril extending from the cell surface (26,31,44). Bioinformatics analysis suggests Aap consists of two distinct domains, the A domain and B domain (28,45).…”

“…Each of these B repeats is composed of 2 subdomains, G5 and E. The G5 unit is the functional component of the B repeat due to its propensity for self association; thus, it coordinates Aap-dependent intercellular interactions (27,30). E spacer regions are located between G5 units and likely prevent misfolding of the protein, a potential consequence of its extended fibrillar nature and repetitive domain architecture (44). Following extracellular export directed by the N-terminal signal sequence, Aap is anchored to the cell wall at the C-terminally located LPDTG sortase recognition motif (28,47).…”