The open-closed transitions within dynamic conformational changes of enzyme loops

Abstract

This review provides a comprehensive overview of the conformational changes between the open and closed states of loop structures within enzyme molecules and their significance in enzyme engineering. The article begins by introducing the developmental background of protein engineering and the structural characteristics of enzymes, with a particular focus on the pivotal role of loops in enzyme activity, substrate specificity, and environmental adaptability. Through instrumental analysis and computational approaches, the molecular mechanisms underlying loop opening and closing are thoroughly examined, and recent practices of loop engineering based on point mutations, directed evolution, and rational design are systematically summarized. The studies indicate that the dynamic changes of loops are central to influencing catalytic efficiency and specificity, and their modification can significantly optimize enzyme performance. Therefore, the in-depth research in this field not only provides novel perspectives for elucidating the dynamic processes of enzymatic catalysis but also paves innovative pathways for optimizing engineering strategies to develop high-performance artificial enzymes and green biomanufacturing technologies.

Keywords

Enzyme / Loop / Open-closed transitions / Conformation

References

[1] Enright AJ, Kunin V, Ouzounis CA. Protein families and TRIBES in genome sequence space. Nucleic Acids Res, 2003, 31(15): 4632-4638

[2] Bergeson AR, Alper HS. Advancing sustainable biotechnology through protein engineering. Trends Biochem Sci, 2024, 49(11): 955-968

[3] Bornscheuer UT, Huisman GW, Kazlauskas RJ, et al.. Engineering the third wave of biocatalysis. Nature, 2012, 485(7397): 185-194

[4] Zoller MJ, Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res, 1982, 10(20): 6487-6500

[5] Chen K, Arnold FH. Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide. Proc Natl Acad Sci U S A, 1993, 90(12): 5618-5622

[6] Stemmer WP. Rapid evolution of a protein in vitro by DNA shuffling. Nature, 1994, 370(6488): 389-391

[7] Bhattacharya R, Rose PW, Burley SK, et al.. Impact of genetic variation on three dimensional structure and function of proteins. PLoS ONE, 2017, 12(3 e0171355

[8] Dey S, Chakrabarti P, Janin J. A survey of hemoglobin quaternary structures. Proteins, 2011, 79(10): 2861-2870

[9] Kendrew JC, Bodo G, Dintzis HM, et al.. A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature, 1958, 181(4610): 662-666

[10] Jisna VA, Jayaraj PB. Protein structure prediction: conventional and deep learning perspectives. Protein J, 2021, 40(4): 522-544

[11] Muduli S, Karmakar S, Mishra S. Conformational dynamics in corynebacterium glutamicum Diaminopimelate epimerase: insights from ligand parameterization, atomistic simulation, and markov state modeling. J Chem Inf Model, 2024, 64(10): 4250-4262

[12] Miyakawa T, Mizushima H, Ohtsuka J, et al.. Structural basis for the Ca(2+)-enhanced thermostability and activity of PET-degrading cutinase-like enzyme from Saccharomonospora viridis AHK190. Appl Microbiol Biotechnol, 2015, 99(10): 4297-4307

[13] Gruninger RJ, Selinger LB, Mosimann SC. Effect of ionic strength and oxidation on the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr. FEBS J, 2008, 275(15): 3783-3792

[14] Montemiglio LC, Gugole E, Freda I, et al.. Point mutations at a key site alter the Cytochrome P450 OleP structural dynamics. Biomolecules, 2021

[15] Gabelli SB, Azurmendi HF, Bianchet MA, et al.. X-ray, NMR, and mutational studies of the catalytic cycle of the GDP-mannose mannosyl hydrolase reaction. Biochemistry, 2006, 45(38): 11290-11303

[16] Johnson TA, Holyoak T. The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function. Biochemistry, 2012, 51(47): 9547-9559

[17] Glaeser RM. How good can cryo-EM become?. Nat Methods, 2016, 13(1): 28-32

[18] Kampjut D, Sazanov LA, The coupling mechanism of mammalian respiratory complex Ⅰ. Science. 2020;(Oct.30 TN.6516): 370.

[19] Faylo JL, Christianson DW. Visualizing transiently associated catalytic domains in assembly-line biosynthesis using cryo-electron microscopy. J Struct Biol, 2021, 213(4 107802

[20] Venkitakrishnan RP, Zaborowski E, McElheny D, et al.. Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle. Biochemistry, 2004, 43(51): 16046-16055

[21] McElheny D, Schnell JR, Lansing JC, et al.. Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. Proc Natl Acad Sci U S A, 2005, 102(14): 5032-5037

[22] Desamero R, Rozovsky S, Zhadin N, et al.. Active site loop motion in triosephosphate isomerase: T-jump relaxation spectroscopy of thermal activation. Biochemistry, 2003, 42(10): 2941-2951

[23] Xu Y, Zheng Y, Fan JS, et al.. A new strategy for structure determination of large proteins in solution without deuteration. Nat Methods, 2006, 3(11): 931-937

[24] Sun J, Voss J, Hubbell WL, et al.. Proximity between periplasmic loops in the lactose permease of Escherichia coli as determined by site-directed spin labeling. Biochemistry, 1999, 38(10): 3100-3105

[25] Roser P, Weisner J, Simard JR, et al.. Direct monitoring of the conformational equilibria of the activation loop in the mitogen-activated protein kinase p38α. Chem Commun (Camb), 2018, 54(85): 12057-12060

[26] Katiki M, Neetu N, Pratap S, et al.. Biochemical and structural basis for Moraxella catarrhalis enoyl-acyl carrier protein reductase (FabI) inhibition by triclosan and estradiol. Biochimie, 2022, 198: 8-22

[27] Hupfeld E, Schlee S, Wurm JP, et al.. Conformational modulation of a mobile loop controls catalysis in the (βα)(8)-barrel enzyme of histidine biosynthesis HisF. JACS Au, 2024, 4(8): 3258-3276

[28] Krishna R, Wang J, Ahern W, et al.. Generalized biomolecular modeling and design with RoseTTAFold All-Atom. Science, 2024, 384(6693 eadl2528

[29] Mandell DJ, Coutsias EA, Kortemme T. Sub-angstrom accuracy in protein loop reconstruction by robotics-inspired conformational sampling. Nat Methods, 2009, 6(8): 551-552

[30] Stein A, Kortemme T. Improvements to robotics-inspired conformational sampling in rosetta. PLoS ONE, 2013, 8(5 e63090

[31] Lyu J, Kapolka N, Gumpper R, et al.. AlphaFold2 structures guide prospective ligand discovery. Science, 2024, 384(6702 eadn6354

[32] Wang J, Wang W, Shang Y. Protein loop modeling using AlphaFold2. IEEE ACM Trans Comput Biol Bioinform, 2023, 20(5): 3306-3313

[33] Zhou J. Huang M, navigating the landscape of enzyme design: from molecular simulations to machine learning. Chem Soc Rev, 2024, 53(16): 8202-8239

[34] Watson JL, Juergens D, Bennett NR, et al.. De novo design of protein structure and function with RFdiffusion. Nature, 2023, 620(7976): 38

[35] Chaves EJF, Coêlho DF, Cruz CHB, et al.. Structure-based computational design of antibody mimetics: challenges and perspectives. FEBS Open Bio, 2025, 15(2223-235

[36] Hollingsworth SA, Dror RO. Molecular dynamics simulation for all. Neuron, 2018, 99(6): 1129-1143

[37] Harish MS, Patra PK, Temperature and its control in molecular dynamics simulations. Molecular Simulation. 2021;(9):

[38] Klein CD. Bachelier A, molecular modeling and bioinformatical analysis of the antibacterial target enzyme MurA from a drug design perspective. J Comput Aided Mol Des, 2006, 20(10–11): 621-628

[39] Mehdi S, Smith Z, Herron L, et al.. Enhanced sampling with machine learning. Annu Rev Phys Chem, 2024, 75(1): 347-370

[40] Xi Z, Rao J, Zhang X et al., Deciphering the Key Loop: Enhancing l-Threonine Transaldolase’s Catalytic Potential.

[41] Biarnés X, Pietrucci F, Marinelli F, et al.. METAGUI. A VMD interface for analyzing metadynamics and molecular dynamics simulations. Comput Phys Commun, 2012, 183(1): 203-211

[42] Liao Q, Kulkarni Y, Sengupta U, et al.. Loop motion in triosephosphate isomerase is not a simple open and shut case. J Am Chem Soc, 2018, 140(46): 15889-15903

[43] Guallar V, Wallrapp F, Mapping protein electron transfer pathways with QM/MM methods. J R Soc Interface. 2008;5 Suppl 3(Suppl 3): S233–239.

[44] van den Bedem H. Fraser JS, integrative, dynamic structural biology at atomic resolution–it's about time. Nat Methods, 2015, 12(4): 307-318

[45] Fenwick RB, van den Bedem H, Fraser JS, et al.. Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR. Proc Natl Acad Sci U S A, 2014, 111(4): E445-454

[46] Li M, You C, Guo F, et al.. Reaction mechanism and regioselectivity of uridine diphosphate glucosyltransferase RrUGT3: a combined experimental and computational study. Catal Sci Technol, 2024, 14(17 14

[47] Zinovjev K, Guénon P, Ramos-Guzmán CA, et al.. Activation and friction in enzymatic loop opening and closing dynamics. Nat Commun, 2024, 15(1 2490

[48] Drago VN, Phillips RS, Kovalevsky A. Universality of critical active site glutamate as an acid-base catalyst in serine hydroxymethyltransferase function. Chem Sci, 2024, 15(32): 12827-12844

[49] Shukla H, Kumar V, Singh AK, et al.. Insight into the structural flexibility and function of Mycobacterium tuberculosis isocitrate lyase. Biochimie, 2015, 110: 73-80

[50] Waki T, Imaizumi R, Uno K, et al.. Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: coenzyme A-induced alteration of product specificity. Biochem Biophys Res Commun, 2024, 718 150080

[51] Buchbinder JL, Fletterick RJ. Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding. J Biol Chem, 1996, 271(37): 22305-22309

[52] Zhang Y, An J, Yang GY, et al.. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426. PLoS ONE, 2015, 10(2 e0115130

[53] Dugdale ML, Vance ML, Wheatley RW, et al.. Importance of Arg-599 of β-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop. Biochem Cell Biol, 2010, 88(6): 969-979

[54] Suzuki K, Maeda S, Morokuma K. Roles of closed- and open-loop conformations in large-scale structural transitions of l-lactate dehydrogenase. ACS Omega, 2019, 4(1): 1178-1184

[55] Zhou J, Han T, Ahmad S, et al.. Origin of the enantioselectivity of alcohol dehydrogenase. Phys Chem Chem Phys, 2023, 25(45): 31292-31300

[56] Shimozawa Y, Himiyama T, Nakamura T, et al.. Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus. Protein Eng Des Sel, 2021

[57] Yang J, Zhang X, Sun Q, et al.. Modulation of the catalytic activity and thermostability of a thermostable GH7 endoglucanase by engineering the key loop B3. Int J Biol Macromol, 2023, 248 125945

[58] Sun J, Boyle AL, Brünle S, et al.. A low-barrier proton shared between two aspartates acts as a conformational switch that changes the substrate specificity of the β-lactamase BlaC. Int J Biol Macromol, 2024, 278(Pt 2 134665

[59] Quraishi O, Nägler DK, Fox T, et al.. The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide. Biochemistry, 1999, 38(16): 5017-5023

[60] Baca AM, Sirawaraporn R, Turley S, et al.. Crystal structure of Mycobacterium tuberculosis 7,8-dihydropteroate synthase in complex with pterin monophosphate: new insight into the enzymatic mechanism and sulfa-drug action. J Mol Biol, 2000, 302(5): 1193-1212

[61] Yeh CY, Izaguirre JA, Greisman JB, et al.. A conserved local structural motif controls the kinetics of PTP1B catalysis. J Chem Inf Model, 2023, 63(13): 4115-4124

[62] Ouedraogo D, Souffrant M, Yao XQ, et al.. Non-active site residue in loop L4 alters substrate capture and product release in d-arginine dehydrogenase. Biochemistry, 2023, 62(5): 1070-1081

[63] Cui DS, Lipchock JM, Brookner D, et al.. Uncovering the molecular interactions in the catalytic loop that modulate the conformational dynamics in protein tyrosine phosphatase 1B. J Am Chem Soc, 2019, 141(32): 12634-12647

[64] Zhao Z, Shen X, Chen S, et al.. Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase. Elife, 2023

[65] Bhattacharjee K. Gopi S, Naganathan AN, a disordered loop mediates heterogeneous unfolding of an ordered protein by altering the native ensemble. J Phys Chem Lett, 2020, 11(16): 6749-6756

[66] Chen CL, Paul LN, Mermoud JC, et al.. Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase. Nat Commun, 2020, 11(1 3969

[67] Shen R, Meng Y, Roux B, et al.. Mechanism of voltage gating in the voltage-sensing phosphatase Ci-VSP. Proc Natl Acad Sci U S A, 2022, 119(44 e2206649119

[68] Liu L, Kohout SC, Xu Q, et al.. A glutamate switch controls voltage-sensitive phosphatase function. Nat Struct Mol Biol, 2012, 19(6): 633-641

[69] Sruthi CK, Meher P, De BAG. Amino acid impact factor. PLoS ONE, 2018, 13(6 e0198645

[70] Li R, Xu M, Xu M, et al.. Semirational engineering of a distal loop region to enhance the catalytic activity and stability of leucine dehydrogenase. J Agric Food Chem, 2025, 73(5): 2961-2969

[71] Savino S, Desmet T, Franceus J. Insertions and deletions in protein evolution and engineering. Biotechnol Adv, 2022, 60 108010

[72] Hoque MA, Zhang Y, Chen L, et al.. Stepwise loop insertion strategy for active site remodeling to generate novel enzyme functions. ACS Chem Biol, 2017, 12(5): 1188-1193

[73] Qi Y, Zhu J, Zhang K, et al.. Recent development of directed evolution in protein engineering. Synthetic Biol J., 2022, 3(6): 1081-1108

[74] Porter JL. Rusli RA, Ollis DL, directed evolution of enzymes for industrial biocatalysis. ChemBioChem, 2016, 17(3): 197-203

[75] Xu Y, Li F, Xie H, et al.. Directed evolution of Escherichia coli surface-displayed Vitreoscilla hemoglobin as an artificial metalloenzyme for the synthesis of 5-imino-1,2,4-thiadiazoles. Chem Sci, 2024, 15(20): 7742-7748

[76] Jacquet P, Billot R, Shimon A et al., Changes in active site loop conformation relate to the transition toward a novel enzymatic activity. bioRxiv. 2023:

[77] Zhou TS, Shen JD, Li XY, et al.. A highly stereoselective and efficient Biocatalytic synthesis of Chiral Syn-Aryl β-Hydroxy α-Amino esters. ACS Catal, 2024, 14(20): 12

[78] Reetz MT. A breakthrough in protein engineering of a glycosyltransferase. Green Synth Catal, 2021

[79] Mou K, Guo Y, Xu W, et al.. Stereodivergent protein engineering of fatty acid photodecarboxylase for light-driven kinetic resolution of sec-alcohol oxalates. Angew Chem Int Ed Engl, 2024, 63(10e202318374

[80] Yang K, Huang Y, Amanze C, et al.. Computer-aided flexible loops engineering of glutamate dehydrogenase for asymmetric synthesis of chiral pesticides l-phosphinothricin. J Agric Food Chem, 2024, 72(4424643-24654

[81] Tiwari MK, Singh R, Singh RK, et al.. Computational approaches for rational design of proteins with novel functionalities. Comput Structural Biotechnol J., 2012, 2(3): e201204002

[82] Schlick T, Molecular Modeling and Simulation: An Interdisciplinary Guide. 2010.

[83] Yue X, Li Y, Wei M, et al.. Rational redesign of the loop dynamics of carbonyl reductase LfSDR1 to improve the stereoselectivity for asymmetric synthesis of bulky chiral alcohols. Int J Biol Macromol, 2024, 274(Pt 2133345

[84] Casadevall G, Casadevall J, Duran C, et al.. The shortest path method (SPM) webserver for computational enzyme design. Protein Eng Des Sel, 2024, 37: gzae005

[85] Duran C, Kinateder T, Hiefinger C, et al.. Altering active-site loop dynamics enhances standalone activity of the tryptophan synthase alpha subunit. ACS Catal, 2024, 14(2216986-16995

[86] Corbella M. Pinto GP, Kamerlin SCL, Loop dynamics and the evolution of enzyme activity. Nat Rev Chem, 2023, 7(8536-547

[87] Kim IV, Navarrete C, Grau-Bové X, et al.. Chromatin loops are an ancestral hallmark of the animal regulatory genome. Nature, 2025, 642(8069): 1097-1105

[88] Ramanathan A. Agarwal PK, evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis. PLoS Biol, 2011, 9(11 e1001193

[89] Jumper J, Evans R, Pritzel A, et al.. Highly accurate protein structure prediction with AlphaFold. Nature, 2021, 596(7873): 583-589

[90] Goodson K A, Wang Z, Haeusler A R, et al. LacI-DNA-IPTG loops: equilibria among conformations by single-molecule FRET. J Phys Chem B. 2013;117(16):4713–22.

Funding

National Institute of Nursing Research(2023YFA0914500)

Institute for National Strategic Studies(32201034)

Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, P. R. China(2023T020)

Fundamental Research Funds for the Central Universities of Beijing University of Chemical Technology(JUSRP124019)

FP7 Ideas: European Research Council(KLIB-KF202402)

Basic and Applied Basic Research Foundation of Guangdong Province(BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University