Surface sites for engineering allosteric control in proteins

Abstract

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites.

Fig. 1

A design concept for allosteric communication. (A) The SCA computational method identifies networks of statistically interacting amino acids in proteins (blue arrows). These networks often link primary functional sites (yellow) with distant surface positions (red) through the core (blue). This motivates the idea of functionally coupling the two proteins (denoted “input” and “output” modules) through linkage at the predicted allosteric sites. (B) A slice through the structure of a complex between the PDZ domain of the cell polarity protein Par6 (cyan surface) with its allosteric regulator, the Cdc42 G protein (white surface) shows an example of SCA network linkage in a natural protein-protein interaction. SCA residues [in CPK representation and colored as in (A)] constitute a contiguous network linking the site of nucleotide exchange on Cdc42 with the ligand-binding pocket of the PDZ domain (both in yellow) through specific residues at the allosteric interface (red).

Fig. 2

Design principle of the PAS-DHFR chimera. (A and B) Surface-exposed SCA sites shown on the LOV2 PAS domain [(A), PDB code 2VOU] and E. coli DHFR [(B), PDB code 1RX2] shown in four successive rotations of each molecule. As in Fig. 1A, SCA network positions are colored yellow (within 5 Å of substrate), red (surface-exposed), or blue (buried). A residue is considered buried if its fractional solvent-accessible surface area is <0.1. In the core PAS domain, this analysis reveals two surface-exposed nonsubstrate proximal SCA sites: (i) the N- and C-terminal regions that, in LOV2, mediate light-dependent interaction with the Jα and N-terminal helical extensions, and (ii) the α3/β4-β5 region (see text and fig. S4). In DHFR, SCA reveals a network (fig. S5) that relates the enzyme active site to a specific distant surface loop (βF-βG, site A, in red). The experiment is to insert the LOV2 domain into several DHFR positions at site A and at a control surface (site B, αC-βE loop).

Fig. 3

Light-dependent catalysis in a LOV2-DHFR chimera. (A and B) Schematics of the chimeric constructs in sites A and B and associated hydride transfer rates (k_hyd) carried out under single-turnover conditions. Data are measured either upon dark adaptation (black bars) or immediately after a 5-min exposure to intense white light (white bars) (26). The A120-noJ chimera lacks the C-terminal Jα helix (a major site of light-dependent conformational change in LOV2), and the A120-C450S chimera carries a point mutation that locks LOV2 in the dark state. No site B chimeras show light-dependent catalysis, but one site A chimera (A120) shows a modest but clear increase in k_hyd upon light activation. (C) Dark (black curves) and light-activated (red curves) absorbance spectra in the A120 and A120-C450S chimeras show the characteristic 447-nm peak of the FMN chromophore in the dark state of LOV2; in A120 alone, light activation shows the expected transition to the 390 nm–absorbing lit-state species. (D) The kinetics of dark recovery of the catalytic rate follows a single-exponential process (red curve), which closely mimics that kinetics of recovery of the 447 nm–absorbing dark state of LOV2 (blue curve). In all panels, error bars indicate SD.

Fig. 4

Light dependence of product off-rate (A) and cofactor binding (B) in the A120 chimera. In (A), the product dissociation rate (k_off, H₄F) shows a small light dependence in A120 (factor of ~1.3) that is abrogated in the background of the dark-locked C450S mutation. (B), Cofactor binding (K_d, NADPH) shows no light dependence. In both assays, the overall effect of LOV2 domain insertion between positions 120 and 121 in the βF-βG loop is similar to that of the point mutation G121V. Error bars indicate SD.