Revealing the Effect of Threonine on the Binding Ability of Antifreeze Proteins with Ice Crystals by Free-energy Calculations

doi:10.7503/cjcu20210838

Abstract

Abstract:

The effect of the threonine content at the ice-binding site（IBS） of moderately active Lolium perenne antifreeze protein（LpAFP） on its binding ability to ice crystals was investigated， employing molecular dynamics simulations and free-energy calculations. A series of mutants of LpAFP were constructed by gradually increasing the amount of threonine on the IBS， including a mutation of eleven sites to make each β-strand have a Thr-x-Thr motif（x is a nonconserved amino acid， mostly a hydrophobic residue）. An importance-sampling algorithm， WTM-eABF， was used to explore the free-energy profile characterizing the adsorption process for LpAFP and its different mutants. WTM-eABF merges the key features of well-tempered metadynamics?“flooding valleys” and an extended Lagrangian variant of adaptive biasing force?“shaving barriers”（WTM-eABF）， which greatly improves the sampling efficiency of the algorithm. The free-energy calculation results indicate that the higher the threonine content of the IBS， the energetically more favorable for binding of AFPs to ice crystals. The mutant with a repeating sequence Thr-x-Thr motif on its flat β-sheet region has the strongest binding affinity to ice. Further analysis showed that the higher the content of threonine， the more ordered water molecules around the IBS， the longer the existence of the anchored clathrate water， and the more stable hydrogen bond network between the IBS and ice surface， thus improving the association strength of AFPs with ice. Overall， increasing the content of threonine residues could significantly enhance the binding ability to ice， which constitutes an approach to improve the antifreeze activity of antifreeze proteins with moderate activity.

Key words: Antifreeze protein, Free-energy calculation, Ice crystal, Binding affinity, Hydrogen bond

CLC Number:

O641

TrendMD:

CUI Shaoli, ZHANG Weijia, SHAO Xueguang, CAI Wensheng. Revealing the Effect of Threonine on the Binding Ability of Antifreeze Proteins with Ice Crystals by Free-energy Calculations[J]. Chem. J. Chinese Universities, 2022, 43(3): 20210838.

Figures/Tables 6

Fig.1 Schematic representation of TmAFP(PDB ID: 1EZG) aligned to the basal plane of ice(A), crystal structures of TmAFP and LpAFP(PDB ID: 3ULT)(B)(A) Red spheres indicate oxygen atoms in the ice lattice and within the hydroxyl groups of threonine. Horizontal dotted red lines highlight the near?perfect two?dimensional match of the interstrand threonine residues to the basal plane. Vertical dotted red lines highlight the match of the intrastrand threonine residues to a section of the basal plane. (B) TmAFP and LpAFP, with 0.48 nm between adjacent β?strands and 0.70 and 0.68 nm, between residues separated by two positions on the same β?strand, respectively. AFP backbones and IBSs are shown in cartoon and stick representations, respectively. The front view is the ice?binding face of the AFPs.

Table 1 Details of the molecular assemblies investigated in this study

Molecular assembly	Number of atom	Box/nm³	Simulation time/ns
LpAFP	57223	7.1×8.8×7.3	400
Mutant1 ^a	52610	7.1×8.1×7.3	400
Mutant2 ^b	52578	7.1×8.1×7.3	400
Mutant3 ^c	52545	7.1×8.1×7.3	400
Mutant4 ^d	52516	7.1×8.1×7.3	400

Fig.2 LpAFP/mutant?ice?water assemblies for MD(A), schematic diagram of the a?axis of ice(B) and LpAFP/mutant?ice?water assemblies for free?energy calculations(C)AFP backbones are shown in cartoon. (A) The distance between the IBS and the basal plane of ice crystals is 1.0 nm. (C) d denotes the projection along the y direction of Cartesian space of the vector connecting the center of mass of the oxygen atoms of the ice and that of the AFP.

Fig.3 Radial distribution function of water(oxygen atoms) around the IBS of LpAFP and its mutants obtained from MD simulations(A), hydrogen bond lifetime correlation functions between any water molecules and amino acid residues at the IBS for wild?type LpAFP and its mutants adsorbed on ice crystals(B)

Fig.4 Free?energy profiles characterizing the adsorption process for LpAFP, Mutant 2 and Mutant 4(A), free?energy profiles at different simulation times for the LpAFP(B), Mutant 2(C) and Mutant 4(D)Inset of (A): water molecules around threonine form water cages. The results show that the calculations have converged within 800 ns.

Fig.5 Hydrogen bond structures of water molecules within 0.5 nm below the IBS of LpAFP(A), Mutant 2(B) andMutant 4(C) adsorbed on ice crystalsCW is shown in vdW representation(oxygen atoms shown in yellow).

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