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.
"The resulting closer packing of the third helix in the core of PSD-1 may also explain its higher melting temperature (85°C) compared to G148-ABD (75°C), which has an isoleucine in this position . Data on the dynamics of PSD-1 also demonstrate that, since PSD-1 is less flexible than G148-ABD and at the same time binds phylogenetically diverse albumins more tightly, broad species specificity can be achieved without an increased backbone flexibility . Previous studies have proposed that the backbone flexibility of G148-ABD is the reason behind its broader specificity compared to ALB8-GA . "
[Show abstract][Hide abstract] ABSTRACT: The albumin-binding domain is a small, three-helical protein domain found in various surface proteins expressed by gram-positive bacteria. Albumin binding is important in bacterial pathogenesis and several homologous domains have been identified. Such albumin-binding regions have been used for protein purification or immobilization. Moreover, improvement of the pharmacokinetics, through the non-covalent association to albumin, by fusing such domains to therapeutic proteins has been shown to be successful. Domains derived from streptococcal protein G and protein PAB from Finegoldia magna, which share a common origin and therefore represent an interesting evolutionary system, have been thoroughly studied structurally and functionally. Their albumin-binding sites have been mapped and these domains form the basis for a wide range of protein engineering approaches. By substitution-mutagenesis they have been engineered to achieve a broader specificity, an increased stability or an improved binding affinity, respectively. Furthermore, novel binding sites have been incorporated either by replacing the original albumin-binding surface, or by complementing it with a novel interaction interface. Combinatorial protein libraries, where several residues have been randomized simultaneously, have generated a large number of new variants with desired binding characteristics. The albumin-binding domain has also been utilized to explore the relationship between three-dimensional structure and amino acid sequence. Proteins with latent structural information built into their sequence, where a single amino acid substitution shifts the equilibrium in favor of a different fold with a new function, have been designed. Altogether, these examples illustrate the versatility of the albumin-binding domain as a scaffold for protein engineering.
Computational and Structural Biotechnology Journal 03/2013; 6(7):e201303009. DOI:10.5936/csbj.201303009
"This was done by adding increasing concentrations of IgG or HSA to a known non-binder and measuring the decrease in amide peak intensities. Thus, the viscosity affect of IgG was determined by adding IgG to the non-binder, 15 N-labeled PSD-1, a variant of wild-type G A (He et al., 2006). Similarly, HSA was added to the non-binder, 15 N-labeled wild-type G B , to determine the HSA viscosity affect. "
[Show abstract][Hide abstract] ABSTRACT: While disordered to ordered rearrangements are relatively common, the ability of proteins to switch from one ordered fold to a completely different fold is generally regarded as rare, and few fold switches have been characterized. Here, in a designed system, we examine the mutational requirements for transitioning between folds and functions. We show that switching between monomeric 3α and 4β+α folds can occur in multiple ways with successive single amino acid changes at diverse residue positions, raising the likelihood that such transitions occur in the evolution of new folds. Even mutations on the periphery of the core can tip the balance between alternatively folded states. Ligand-binding studies illustrate that a new immunoglobulin G-binding function can be gained well before the relevant 4β+α fold is appreciably populated in the unbound protein. The results provide new insights into the evolution of fold and function.
"The differences in backbone structure between free and bound GA domains are small with a displacement of the a3-helix of ;1–1.5 A ˚ (He et al. 2006), but these subtle effects appear to lead to changes in albumin affinity that can have a dramatic effect on species specificity. Of further note, the considerable exchange-broadening effects observed for the naturally occurring GA domains ALB8–GA and G148–GA3 with RSA were mitigated by using the PSD-1 phage selected domain, even though the domain was originally selected for optimal binding to other related albumins (HSA and GPSA). "
[Show abstract][Hide abstract] ABSTRACT: Protein G-related albumin-binding (GA) modules occur on the surface of numerous Gram-positive bacterial pathogens and their presence may promote bacterial growth and virulence in mammalian hosts. We recently used phage display selection to evolve a GA domain, PSD-1 (phage selected domain-1), which tightly bound phylogenetically diverse albumins. With respect to PSD-1's broad albumin binding specificity, it remained unclear how the evolved binding epitope compared to those of naturally occurring GA domains and whether PSD-1's binding mode was the same for different albumins. We investigate these questions here using chemical shift perturbation measurements of PSD-1 with rabbit serum albumin (RSA) and human serum albumin (HSA) and put the results in the context of previous work on structure and dynamics of GA domains. Combined, these data provide insights into the requirements for broad binding specificity in GA-albumin interactions. Moreover, we note that using the phage-optimized PSD-1 protein significantly diminishes the effects of exchange broadening at the binding interface between GA modules and albumin, presumably through stabilization of a ligand-bound conformation. The employment of artificially evolved domains may be generally useful in NMR structural studies of other protein-protein complexes.
Protein Science 08/2007; 16(7):1490-4. DOI:10.1110/ps.072799507 · 2.85 Impact Factor
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