Engineering Allostery into Proteins

Abstract

Our ability to engineer protein structure and function has grown dramatically over recent years. Perhaps the next level in protein design is to develop proteins whose function can be regulated in response to various stimuli, including ligand binding, pH changes, and light. Endeavors toward these goals have tested and expanded on our understanding of protein function and allosteric regulation. In this chapter, we provide examples from different methods for developing new allosterically regulated proteins. These methods range from whole insertion of regulatory domains into new host proteins, to covalent attachment of photoswitches to generate light-responsive proteins, and to targeted changes to specific amino acid residues, especially to residues identified to be important for relaying allosteric information across the protein framework. Many of the examples we discuss have already found practical use in medical and biotechnology applications.

Keywords: Allostery; Amino acid network; Covalent modification; Domain insertion; Energy landscape; Protein engineering; Protein regulation.

Fig. 15.1

Frameworks for understanding allostery. (a) In the free energy landscape model, proteins are considered to be ensembles of different conformations. Local minima represent different conformations, with their free energies determining relative populations, and free energy barriers determining exchange rates between conformations. Allosteric inhibitors and activators can change the topology of the energy landscape by altering populations or the kinetics of exchange between conformations. (b) The network model places emphasis on correlated structural dynamics of amino acid residues being responsible for allosteric effects. A perturbation at one site can have an effect on a surrounding cluster of amino acids, which then can propagate through the amino acid residue interaction network to effect changes at a distant active site or other binding site. The network signals travel through highly connected hub residues, which may be important engineering points to change protein function and regulation

Fig. 15.2

Methods of rational domain insertion. (a) Homologous proteins with catalytic domains are often structurally similar enough that regulatory domains sensitive to different effectors can be swapped or added to homologs without a regulatory domain, as in the above example where the blue regulatory domain is spliced onto the orange catalytic domain. (b) In cases where the protein with the desired function does not have any allosteric homologs, a conditionally disordered linker that becomes ordered in response to environmental changes or a regulatory domain can be inserted into the middle of a loop in the host protein sequence. In this example, the green enzymatic domain is inactive when the yellow regulatory domain is in its open apo conformation. Once the regulatory domain binds its effector ligand, it adopts a conformation that allows the enzymatic domain to adopt its active conformation

Fig. 15.3

Examples of allosteric enzymes engineered through domain insertion. (A) ^PfuACT^TmaDAH7PS with Tyr (yellow) bound to the ACT domain (magenta) with the DAH7PS domain in slate (PDB: 4GRS) [16]. ^PfuACT^TmaDAH7PS is typically depicted as a homotetramer. It should be noted that only one subunit is shown here for clarity. (B) DHFR-LOV2 with LOV2 domain inserted at residue 120 of DHFR (PDBs: 1RX2, 2V0U) [59]. Upon irradiation with light, C450 covalently bonds to the flavin cofactor in the LOV2 domain. The changes in conformation and dynamics propagate through both domains. (C) The MBP-BLA construct RG13 generated by random domain insertion of BLA (red) into MBP (blue) (PDB: 4DXB) [48]

Fig. 15.4

Methods of random domain insertion. Details on the major steps of creating gene insertion libraries are shown in brief

Fig. 15.5

Covalent modification of proteins to control allostery. (a) Conjugation of spiropyran to a Cys residue and isomerization to the merocyanine form. (b) Conjugation of the bifunctional azobenzene derivative 4,4′-bis (maleimido)azobenzene to two cysteine residues and isomerization between trans and cis forms. (c) Conjugation of the bifunctional azobenzene derivative 3,3′-bis (sulfonato)-4,4′-bis (chloroacetamido)azobenzene (BSBCA) to two Cys residues and isomerization between trans and cis forms. (d) Isomerization of the genetically encoded azobenzene derivative AzoPhe. (e) Covalent modification of a Lys residue by quinazolin-4 (3H)-one hydroxamic ester. (f) Covalent modification of PvuII with azomal (PDB: 1NI0), adapted from Schierling et al. [108]. Active site residues (cyan) are disrupted upon isomerization of azomal to the trans form

Fig. 15.6

pH-dependent modulation of the NP4 protein. Closed (top) and open (bottom) conformations of NP4. Protonation of Asp30 stabilizes the closed conformation, while deprotonation stabilizes the open conformation. (Reprinted with permission from Di Russo, N. V.; Martí, M. A.; Roitberg, A. E. Underlying Thermodynamics of PH-Dependent Allostery. J. Phys. Chem. B 2014, 118 (45), 12,818–12,826. Copyright 2014 American Chemical Society)

Fig. 15.7

The RASMM method for identifying dynamically coupled amino acid interaction networks in proteins. Amino acids distal from the active site are identified as “hot spots” based on their R₂ relaxation dispersion profiles in the apo and substrate-bound states. Residues that are linked allosterically to the active site are expected to show a measurable change in R₂ relaxation dispersion behavior in the presence of substrate. To identify the global communications networks, single site mutations of “hot spot” residues are generated and global R₂ relaxation dispersion profiles are examined. Reprinted from Structure, Volume 25 Issue 2, MJ Holliday, C Camilloni, GS Armstrong, M Vendruscolo, EZ Eisenmesser. Networks of Dynamic Allostery Regulate Enzyme Function, 276–286, Copyright (2017), with permission from Elsevier

Fig. 15.8

Engineering allostery “out of” the beta subunit of tryptophan synthase. (a) A crystal structure of the COMM domain of βTS representing the differences in the open/closed conformations through the catalytic cycle. The “extended” state represents the open conformation. As the nreaction proceeds, the COMM domain enters the fully closed conformation (E (A-A)). (b) A theoretical reaction coordinate illustrating the energy levels of the βTS intermediates. The engineered beta subunit (PfTrp^B2B9) creates a decrease in energy of the product bound state. This results in a more efficient enzyme without the presence of the allosteric effector (αTS). Reprinted with permission from Buller, A. R. et al. Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble. J. Am. Chem. Soc. 140, 7256–7266 (2018). Copyright 2018 American Chemical Society