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. 2020 Apr 3:10.1088/1478-3975/ab8697.
doi: 10.1088/1478-3975/ab8697. Online ahead of print.

The physical basis and practical consequences of biological promiscuity

Shelley D Copley  1
Affiliations

The physical basis and practical consequences of biological promiscuity

Shelley D Copley. Phys Biol. .

Abstract

Proteins interact with metabolites, nucleic acids, and other proteins to orchestrate the myriad catalytic, structural and regulatory functions that support life from the simplest microbes to the most complex multicellular organisms. These molecular interactions are often exquisitely specific, but never perfectly so. Adventitious "promiscuous" interactions are ubiquitous due to the thousands of macromolecules and small molecules crowded together in cells. Such interactions may perturb protein function at the molecular level, but as long as they do not compromise organismal fitness, they will not be removed by natural selection. Although promiscuous interactions are physiologically irrelevant, they are important because they can provide a vast reservoir of potential functions that can provide the starting point for evolution of new functions, both in nature and in the laboratory.

Keywords: enzyme evolution; evolution; moonlighting; promiscuity; protein dynamics; protein-protein interaction.

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Figures

Figure 1.
Figure 1.
Substrate profiling of esterases (horizontal axis) against a panel of 96 chemically diverse esters (vertical axis). Colored blocks denote activity against a particular substrate. The colors indicate the family to which each enzyme belongs. FI, FII, FIV, FV, FVI and FVII, protein families with an α/β hydrolase fold but different conserved sequence motifs; FVIII, serine β-lactamase-like fold; CE, carbohydrate esterase family; C-C MCPH, carbon-carbon meta cleavage product hydrolase family; cyclase-like esterase family, cyclase-like protein from the amidohydrolase superfamily. Modified with permission from ACS Chem Biol. 2018;13(1):225-34 (2). (Further permissions related to the material excerpted should be directed to the American Chemical Society.)
Figure 2.
Figure 2.
The E. coli cytoplasm is crowded and dynamic. (a) The 51 macromolecules that were included in the simulation. (b) A snapshot of the simulation. Reproduced with permission from PLoS Comput Biol. 2010;6(3):e1000694 (19).
Figure 3.
Figure 3.
Interactions between cations and the pi systems of aromatic rings. (a) The amino group of carbamoyl choline interacts with the aromatic rings of Tyr192 and Trp143 in acetylcholine binding protein. (b) The aromatic system of benzo[a]pyrene interacts with positive charges of the light-chain Lys89 and the heavy-chain Arg95 in monoclonal antibody 4D5. Reproduced with permission from J Agric Food Chem 66(13):3315-23, Liang Z, Li QX, pi-Cation interactions in molecular recognition: Perspectives on pharmaceuticals and pesticides. Copyright 2018 American Chemical Society (38).
Figure 4.
Figure 4.
Part of the protein-protein interaction surface between SoxY (orange), SoxZ (yellow) and SoxB (grey), which are involved in thiosulfate oxidation in Thermus thermophilus (41). This interaction is relatively weak; strong protein-protein interactions involve larger surface areas. Reproduced with permission from Proc Natl Acad Sci USA. 2015; 112(52):E7166-75. Grabarczyk DB, Chappell PE, Johnson S, Stelzl LS, Lea SM, Berks BC. Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle.
Figure 5.
Figure 5.
Substrates for the promiscuous N-acyl amino acid racemase activity of o-succinylbenzoate synthase are malpositioned in the active site. (a) The native and promiscuous reactions catalyzed by the enzyme. The proton that is abstracted by the active site base is highlighted in red. (b) The positions of OSB (an analog for the native substrate) and three promiscuous substrates relative to the active-site divalent cation (circles) (42). Diamonds mark the carbon from which a proton is abstracted in the first step of the reaction. Reproduced with permission from Biochemistry 2004;43(19):5716-27 Thoden JB, Taylor Ringia EA, Garrett JB, Gerlt JA, Holden HM, Rayment I. Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis. Copyright 2004 American Chemical Society.
Figure 6.
Figure 6.
Structures of resveratrol-protein complexes. (a) transthyretin, a thyroxine transport protein (PDB 5CR1); (b) cardiac troponin C (PDB 2L98); (c) myosin 2 heavy chain motor domain (PDB 3MNQ); (d) tyrosyl tRNA synthetase (PDB 4Q93). Regions of secondary structure that contribute resveratrol binding residues are highlighted in green. Reprinted with permission from Front Pharmacol. 2018;9:1201.
Figure 7.
Figure 7.
Proteins exist in a dynamic equilibrium between substates. Amino acid side-chain motions and loop motions on the ps-ns timescale generate multiple substates within structural ensembles. Slower motions correspond to larger conformational changes between ensembles. (50) Reprinted from Biochim Biophys Acta, 1814(8), Kleckner IR, Foster MP. An introduction to NMR-based approaches for measuring protein dynamics, 942-68. Copyright 2011, with permission from Elsevier.
Figure 8.
Figure 8.
Conformational changes in the active site of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase facilitate substrate binding and catalysis. (a) the reaction mechanism (56). Reprinted with permission from Levsh O, Chiang YC, Tung CF, Noel JP, Wang Y, Weng JK. Dynamic conformational states dictate selectivity toward the native substrate in a substrate-permissive acyltransferase. Biochemistry 2016;55(45):6314-26. Copyright 2016 American Chemical Society. (b) catalytic residues in the active site adopt different conformations in the presence of the product, p-coumaroylshikimate, which serves as a substrate analog. Blue, apoenzyme (PDB 5KJS); tan, enzyme complexed with p-coumaroylshikimate (PDB 5KJU).
Figure 9.
Figure 9.
Models for conformational changes associated with ligand binding (62). In the induced fit model, ligand binding changes the conformation of the protein from P1 to P2. In the conformational selection model, the ligand is only able to bind to P2. Reprinted by permission from Springer Nature: Nat Chem Biol. 5(11):789-96 The role of dynamic conformational ensembles in biomolecular recognition. Copyright 2009.
Figure 10.
Figure 10.
The active site of an evolved arylesterase. The side chain of Arg254, which exhibits conformational flexibility in the promiscuous progenitor enzyme, is frozen in a bent conformation that enhances binding of the 2-naphthol hexanoate substrate. Reproduced with permission from Springer Nature: Nat Chem Biol. 2016;12(11):944-50. Campbell E, Kaltenbach M, Correy GJ, Carr PD, Porebski BT, Livingstone EK, et al. The role of protein dynamics in the evolution of new enzyme function. Copyright 2016.
Figure 11.
Figure 11.
Promiscuous enzymatic reactions. Substrate ambiguity in ProA (14) and ThrB (10); catalytic promiscuity in PcpC (72) and DesII (71). GSH, glutathione; GSSG, glutathione disulfide; SAM, S-adenosylmethionine; 5’-dAdo, 5’-deoxyadenosine.
Figure 12.
Figure 12.
Promiscuous hydrolase activities of metallo-β-lactamase superfamily enzymes vary depending on the active-site metal ion. Arrows indicate the bond that is cleaved during the reaction. (a) Reactions that were assayed for each enzyme. (b) Levels of activity of purified enzymes (top line) and enzymes reconstituted with the indicated metal ions with each of the substrates shown in A. BLA, Bla-L1, Salmonella maltophilia L1 B3 β-lactamase; bla-VIM2, Pseudomonas aeruginosa VIM2 β-lactamase B1 ; rbn, E. coli ribonuclease B1; mph, P. aeruginosa methyl-parathion hydrolase; atsA, Alteromonas carrageenovora arylsulfatase. X indicates the native reaction for each enzyme. Reprinted with permission from ACS Chem Biol. 2015;10(7):1684-93, Baier F, Chen J, Solomonson M, Strynadka NC, Tokuriki N. Distinct metal isoforms underlie promiscuous activity profiles of metalloenzymes. Copyright 2015 American Chemical Society.
Figure 13.
Figure 13.
Tyr416 in the active site of bacteriophage RB69 DNA polymerase (PDB 1IG9) provides a steric gate that minimizes binding of ribonucleotide substrates due to a steric clash with the 2’-hydroxyl. Molecular graphics analysis was performed with UCSF Chimera v. 1.13, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311 (57).
Figure 14.
Figure 14.
Carboxysomes concentrate CO2 and restrict access of O2 to minimize the promiscuous oxygenation reaction catalyzed by RuBisCO. (a) The structure of the carboxysome showing RuBisCO and carbonic anhydrase sequestered within a rigid protein shell. BMC (bacterial microcompartment proteins form the icosahedral shell and encapsulate RuBisCO and carbonic anhydrase. (Kerfeld lab, Creative Commons international license) (b) Interior surface of a hexameric pore made up of the CcmK1 protein that attracts HCO3− to the interior of the carboxysome (91). Blue and red correspond to positive and negative charges, respectively. Reprinted with permission from Photosynth Res., 109(1-3), 21-32, 2011. Kinney JN, Axen SD, Kerfeld CA, Comparative analysis of carboxysome shell proteins.
Figure 15.
Figure 15.
Mechanism for charging of tRNAs with amino acids by aminoacyl tRNA-synthetases (AS). aa, amino acid; aa-AMP, aminoacyl adenylate. Reprinted from Adv Protein Chem Struct Biol 86, Yadavalli SS, Ibba M, Quality control in aminoacyl-tRNA synthesis: its role in translational fidelity, 1-43, 2012 with permission from Elsevier.
Figure 16.
Figure 16.
The double-sieve mechanism for ensuring proper charging of tRNAs (99). The first sieve is the selection for cognate amino acids at the active sites of aminoacyl tRNA synthetases. The second sieve is the selection for improperly charged tRNAs at an editing site. Reprinted with permission from Ling J, Soll D. Severe oxidative stress induces protein mistranslation through impairment of an aminoacyl-tRNA synthetase editing site. Proc Natl Acad Sci U S A. 2010;107(9):4028-33.
Figure 17.
Figure 17.
A promiscuous activity of malate dehydrogenase produces the toxic metabolite L-2-hydroxyglutarate. The metabolite repair enzyme L-2-hydroxyglutarate dehydrogenase converts toxic L-2-hydroxyglutarate to α-ketoglutarate. Red arrows, physiological reactions; blue arrows, promiscuous reactions.
Figure 18.
Figure 18.
Promiscuous activities of glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase (dashed arrows in yellow boxes) lead to production of toxic metabolites (red) that inhibit other enzymes in central carbon metabolism (107). Blue, inhibited enzymes; purple, the metabolite repair enzyme phosphoglycolate phosphatase. Cofactors and co-substrates are not shown for clarity.
Figure 19.
Figure 19.
The Innovation-Amplification-Divergence (IAD) model for evolution of new proteins. A represents the canonical function of the protein and b represents a promiscuous function. Purple indicates alleles encoding a protein that serves both functions.
Figure 20.
Figure 20.
A sequence similarity network of the nitroreductase superfamily (143). The 24,270 proteins in the superfamily are represented by 5,337 nodes, each of which represents a set of proteins sharing >60% sequence identity. The size of a node reflects the number of sequences it represents. Black borders around nodes indicate proteins for which a biochemical function is known. Triangles depict proteins for which a crystal structure is available. Twenty-two major subgroups are colored differently. White nodes with grey borders indicate sequences that do not belong to any of the colored subgroups. Edges between nodes correspond to Hidden Markov Model alignment scores that are more significant than 10−24. Clusters designated by unk have no members with identified functions. Modified from Akiva E, Copp JN, Tokuriki N, Babbitt PC. Evolutionary and molecular foundations of multiple contemporary functions of the nitroreductase superfamily. Proc Natl Acad Sci U S A. 2017;114(45):E9549-E58.
Figure 21.
Figure 21.
The architecture of the enolase superfamily (145). (a) The barrel domain provides catalytic residues, and the capping domain contributes to specificity. (b) Residues that coordinate the active site metal ion and provide catalytic functions protrude into the active site from loops in the barrel domain. Evolutionary tinkering with these residues has led to enzymes that direct the enolate intermediate to different fates (see Figure 22). Reprinted from Gerlt JA, Babbitt PC, Jacobson MP, Almo SC. Divergent evolution in enolase superfamily: strategies for assigning functions. J Biol Chem. 2012; 287(1): 29-34.
Figure 22.
Figure 22.
Reactions catalyzed by members of the mechanistically diverse enolase superfamily. MLE, muconate lactonizing enzyme; MR, mandelate racemase. Each reaction begins with abstraction of a proton from carbon alpha to a carboxylate, but the subsequent steps differ. Reprinted from Gerlt JA, Babbitt PC, Jacobson MP, Almo SC. Divergent evolution in enolase superfamily: strategies for assigning functions. J Biol Chem. 2012; 287(1): 29-34.
Figure 23.
Figure 23.
Divergence of the ligand-binding domain of TetR transcription factors has resulted in proteins that respond to a wide range of structurally diverse ligands. Republished with permission of the American Society of Microbiology from Cuthbertson L, Nodwell JR. The TetR family of regulators. Microbiol Mol Biol Rev. 77(3), 440-75, 2013; permission conveyed through Copyright Clearance Center, Inc.
Figure 24.
Figure 24.
Most organisms that synthesize PLP use a single enzyme complex comprised of Pdx1 and Pdx2. Atom tracing from (154). Image of PLP synthase created with NGL Viewer (AS Rose et al. (2018) NGL viewer: web-based molecular graphics for large complexes. Bioinformatics doi:10.1093/bioinformatics/bty419), and RCSB PDB.
Figure 25.
Figure 25.
The PLP synthesis pathway in E. coli and other γ-proteobacteria. In α-proteobacteria, the function of PdxB is supplied by an unrelated enzyme, PdxR.
Figure 26.
Figure 26.
Different pathways for degradation of nitrotoluene by Pseudomonas sp. 4NT (155) and Mycobacterium Strain HL 4_NT-1 (156, 157) (top) and degradation of pentachlorophenol by Sphingobium chlorophenolicum (158-160) and Rhodococcus chlorophenolicus (161) (bottom) have emerged in bacteria isolated from contaminated sites.
Figure 27.
Figure 27.
A novel four-step bypass pathway patched together from promiscuous enzymes restores PLP synthesis in an evolved strain of E. coli that lacks PdxB. Promiscuous enzymes that have been recruited to serve new functions are highlighted in red. SerA, 3-phosphoglycerate dehydrogenase; SerC, phosphoserine/phosphohydroxythreonine aminotransferase. Cofactors are shown only for the bypass pathway.
Figure 28.
Figure 28.
LovD acquires α-S-methylbutyrate from the polyketide synthase LovF and transfers it to monacolin J in the natural pathway for synthesis of lovastatin in Aspergillus terreus (top). LovD also has a promiscuous ability to react with a thioester of α-dimethylbutyrate and then transfer the α-dimethylbutyrate to monacolin J to produce simvastatin (bottom). Domains of LovF: KS, ketosynthase; AT, acyltransferase; DH, dehydratase; MT, methyltransferase; ER, enoylreductase; KR, ketoreductase; ACP, acyl carrier protein. Modified from Chem Biol, 16(10), Gao X, Xie X, Pashkov I, Sawaya MR, Laidman J, Zhang W, et al. Directed evolution and structural characterization of a simvastatin synthase, 1064-74, Copyright 2009, with permission from Elsevier.
Figure 29.
Figure 29.
A tRNA/tRNA synthetase pair that is orthogonal to the normal translation machinery can incorporate an unnatural amino acid into an elongating protein at a specific position specified by an amber codon (UAG) in the mRNA. Reprinted from Chem Biol, 16(3), Wang Q, Parrish AR, Wang L. Expanding the genetic code for biological studies. 323-36, Copyright 2009, with permission from Elsevier.
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