Low-stringency selection of TEM1 for BLIP shows interface plasticity and selection for faster binders

Abstract

Protein-protein interactions occur via well-defined interfaces on the protein surface. Whereas the location of homologous interfaces is conserved, their composition varies, suggesting that multiple solutions may support high-affinity binding. In this study, we examined the plasticity of the interface of TEM1 β-lactamase with its protein inhibitor BLIP by low-stringency selection of a random TEM1 library using yeast surface display. Our results show that most interfacial residues could be mutated without a loss in binding affinity, protein stability, or enzymatic activity, suggesting plasticity in the interface composition supporting high-affinity binding. Interestingly, many of the selected mutations promoted faster association. Further selection for faster binders was achieved by drastically decreasing the libraryligand incubation time to 30 s. Preequilibrium selection as suggested here is a novel methodology for specifically selecting faster-associating protein complexes.protein-protein interaction | in vitro evolution | association rate | interface plasticity P rotein-protein interactions play important roles in most cellular processes. Proteins interact through chemical and structural complementarity of their mutual binding sites (1). Amino acids found in physical proximity form noncovalent interactions that stabilize the complex (2). Residues not directly involved in binding also play a role in complex formation by stabilizing the association encounter complex and transition state, which determine the interaction's association rate constant (3). Protein interaction interfaces seem to have dual, seemingly contradicting, properties: On one hand, the interface is a unique area (4) on the protein surface that has evolved to bind specific partners (5). Although much is known about the molecular mechanism of binding, de novo design of protein interactions is still a complicated task, requiring in vitro evolution to achieve high-affinity binding (6-8). On the other hand, interfaces seem to be rather tolerant to mutagenesis (2, 9-11), implying that there are many ways to achieve high-affinity binding. The plastic nature of proteinprotein interfaces becomes particularly clear when comparing the conservation levels of transient complex interfaces versus surface and core residues in homologous complexes (12, 13). Here it is shown that core residues are similarly conserved as permanent interaction interface residues but that transient interaction interface residues are hardly more conserved than other surface residues. The relatively low conservation levels of transient interface residues also make it difficult to predict the protein binding sites (contrary to permanent interfaces) (14, 15). In fact, it was suggested that the vast majority of interfacial residues are important only for conformational optimization of the few interacting residues, resulting in variability in their actual identity (16).The interface formed between different β-lactamase proteins and their inhibitor protein BLIP is a good example of the use...

SKEMPI 2.0: an updated benchmark of changes in protein–protein binding energy, kinetics and thermodynamics upon mutation

“…Tidow et al , 2006), locating druggable binding sites (e.g. Stevers et al , 2017) and binding hotspots for drug design (Guo et al , 2014), altering binding kinetics (Cohen-Khait and Schreiber, 2016; Rosenfeld et al , 2017), protein–protein docking (e.g. Epa et al , 2013), and characterizing transition states (e.g.…”

SKEMPI 2.0: An updated benchmark of changes in protein-protein binding energy, kinetics and thermodynamics upon mutation

“…[72]), locating druggable binding sites (e.g. [68]) and binding hotspots for drug design [25], altering binding kinetics [13], [64], protein-protein docking (e.g. [19]), and characterising transition states (e.g.…”

Efficient production of secretory Streptomyces clavuligerus β-lactamase inhibitory protein (BLIP) in Pichia pastoris

“…First, from the point of view of biophysics, the potent interaction between BLIP and TEM-1 β-lactamase makes BLIP an appealing study model for protein–protein interaction. Determinants for the strong binding of BLIP with TEM-1 β-lactamase have been extensively characterized to elucidate the general principles of affinity and specificity in protein–protein interaction (Strynadka et al 1996 ; Selzer et al 2000 ; Zhang and Palzkill 2003 ; Kozer et al 2007 ; Wang et al 2007 ; Cohen-Khait and Schreiber 2016 ). Second, BLIP has its therapeutic value as a proteinaceous β-lactamase inhibitor.…”