Structure, Dynamics, and Stability Variation in Bacterial Albumin Binding Modules:  Implications for Species Specificity^,

Abstract

Protein G-related albumin-binding (GA) modules are frequently expressed on the surfaces of bacterial cells. The limited amino acid sequence variation among GA modules results in structural and functional differences with possible implications for bacterial pathogenesis and host specificity. In particular, the streptococcal G148-GA3 and F. magna ALB8-GA albumin-binding domains exhibit a degree of structural and dynamic diversity that may account for their varied affinities for different species of albumin. To explore the impact of GA module polymorphisms on albumin binding and specificity, we recently used offset recombinant PCR to shuffle seven artificially constructed representatives of the GA sequence space and scan the phage-displayed recombinant domains for mutations that supported binding to the phylogenetically distinct human and guinea pig serum albumins (HSA and GPSA) (Rozak et al. (2006) Biochemistry 45, 3263-3271). Surprisingly, phage selection revealed an overwhelming preference for a single recombinant domain (PSD-1, phage-selected domain-1) regardless of whether the phages were enriched for their abilities to bind one or both of these albumins. We describe here the NMR-derived structure, dynamics, and stability of unbound PSD-1. Our results demonstrate that increased flexibility is not a requirement for broadened specificity, as had been suggested earlier (Johansson et al. (2002) J. Mol. Biol. 316, 1083-1099), because PSD-1 binds the phylogenetically diverse HSA and GPSA even more tightly than G148-GA3 but is less flexible. The structural basis for albumin-binding specificity is analyzed in light of these new results.

GA/GB Fold switching may modulate fatty acid transfer from human serum albumin to bacteria

“…22) was used to build the model of the hypothetical 4b1a form of PSD-1 using the homology modeling program NEST (23). Then, flexible docking simulations were used to dock a Myr molecule onto both the a-helical form of the GA module PSD-1 (PDB code 2FS1) (24) and the modeled 4b1a form. Figure 2 shows the lowest energy complexes obtained using the AutoDock Vina molecular docking software (25).…”

A minimal sequence code for switching protein structure and function

“…NMR Spectroscopy. 15 N-and 13 C/ 15 N-labeled samples were prepared at 0.2-0.3-mM concentrations in 100 mM KPO4 buffer (pH 7.2) containing 10 mM sodium azide and 5-10% D 2O. NMR spectra were collected on a Bruker AVANCE 600 MHz spectrometer fitted with a z axis gradient 1 H/ 13 C/ 15 N triple resonance cryoprobe.…”

NMR structures of two designed proteins with high sequence identity but different fold and function

“…The A52F mutation causes an Ϸ10-fold decrease in HSA-binding affinity (data not shown), which may be partly due to these small structural changes. Previous work showed that the affinity of G A modules for albumins can be affected by relatively small changes in helical arrangement (25). Most core residues are situated similarly in both proteins apart from those in the ␣3-helix that are displaced 2-3 Å from their wild-type PSD-1 positions.…”