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Design of nanobody-based bispecific constructs by in silico affinity maturation and umbrella sampling simulations.

ABSTRACT: Random mutagenesis is the natural opportunity for proteins to evolve and biotechnologically it has been exploited to create diversity and identify variants with improved characteristics in the mutant pools. Rational mutagenesis based on biophysical assumptions and supported by computational power has been proposed as a faster and more predictable strategy to reach the same aim. In this work we confirm that substantial improvements in terms of both affinity and stability of nanobodies can be obtained by using combinations of algorithms, even for binders with already high affinity and elevated thermal stability. Furthermore, in silico approaches allowed the development of an optimized bispecific construct able to bind simultaneously the two clinically relevant antigens TNF-α and IL-23 and, by means of its enhanced avidity, to inhibit effectively the apoptosis of TNF-α-sensitive L929 cells. The results revealed that salt bridges, hydrogen bonds, aromatic-aromatic and cation-pi interactions had a critical role in increasing affinity. We provided a platform for the construction of high-affinity bispecific constructs based on nanobodies that can have relevant applications for the control of all those biological mechanisms in which more than a single antigen must be targeted to increase the treatment effectiveness and avoid resistance mechanisms.

SUBMITTER: Bai Z

PROVIDER: S-EPMC9822835 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Design of nanobody-based bispecific constructs by <i>in silico</i> affinity maturation and umbrella sampling simulations.

Bai Zixuan Z Wang Jiewen J Li Jiaqi J Yuan Haibin H Wang Ping P Zhang Miao M Feng Yuanhang Y Cao Xiangtong X Cao Xiangan X Kang Guangbo G de Marco Ario A Huang He H

Computational and structural biotechnology journal 20221216

Random mutagenesis is the natural opportunity for proteins to evolve and biotechnologically it has been exploited to create diversity and identify variants with improved characteristics in the mutant pools. Rational mutagenesis based on biophysical assumptions and supported by computational power has been proposed as a faster and more predictable strategy to reach the same aim. In this work we confirm that substantial improvements in terms of both affinity and stability of nanobodies can be obta ...[more]

PMID: 36659922

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Project description:IntroductionRiboswitches are cis-acting regulatory RNA elements prevalently located in the leader sequences of bacterial mRNA. An adenine sensing riboswitch cis-regulates adeninosine deaminase gene (add) in Vibrio vulnificus. The structural mechanism regulating its conformational changes upon ligand binding mostly remains to be elucidated. In this open framework it has been suggested that the ligand stabilizes the interaction of the distal "kissing loop" complex. Using accurate full-atom molecular dynamics with explicit solvent in combination with enhanced sampling techniques and advanced analysis methods it could be possible to provide a more detailed perspective on the formation of these tertiary contacts.MethodsIn this work, we used umbrella sampling simulations to study the thermodynamics of the kissing loop complex in the presence and in the absence of the cognate ligand. We enforced the breaking/formation of the loop-loop interaction restraining the distance between the two loops. We also assessed the convergence of the results by using two alternative initialization protocols. A structural analysis was performed using a novel approach to analyze base contacts.ResultsContacts between the two loops were progressively lost when larger inter-loop distances were enforced. Inter-loop Watson-Crick contacts survived at larger separation when compared with non-canonical pairing and stacking interactions. Intra-loop stacking contacts remained formed upon loop undocking. Our simulations qualitatively indicated that the ligand could stabilize the kissing loop complex. We also compared with previously published simulation studies.Discussion and conclusionsKissing complex stabilization given by the ligand was compatible with available experimental data. However, the dependence of its value on the initialization protocol of the umbrella sampling simulations posed some questions on the quantitative interpretation of the results and called for better converged enhanced sampling simulations.

Project description:The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are flexible molecules, and thus, require explicit or implicit incorporation of multiple conformational states into the computational procedure. The astronomical size of the amino acid sequence space further compounds the challenge by requiring predictions to be computed within a short time so that many sequence variants can be tested. In this study, we compare three classes of methods for antibody/antigen (Ab/Ag) binding affinity calculations: (i) a method that relies on the physical separation of the Ab/Ag complex in equilibrium molecular dynamics (MD) simulations, (ii) a collection of 18 scoring functions that act on an ensemble of structures created using homology modeling software, and (iii) methods based on the molecular mechanics-generalized Born surface area (MM-GBSA) energy decomposition, in which the individual contributions of the energy terms are scaled to optimize agreement with the experiment. When applied to a set of 49 antibody mutations in two Ab/HIV gp120 complexes, all of the methods are found to have modest accuracy, with the highest Pearson correlations reaching about 0.6. In particular, the most computationally intensive method, i.e., MD simulation, did not outperform several scoring functions. The optimized energy decomposition methods provided marginally higher accuracy, but at the expense of requiring experimental data for parametrization. Within each method class, we examined the effect of the number of independent computational replicates, i.e., modeled structures or reinitialized MD simulations, on the prediction accuracy. We suggest using about ten modeled structures for scoring methods, and about five simulation replicates for MD simulations as a rule of thumb for obtaining reasonable convergence. We anticipate that our study will be a useful resource for practitioners working to incorporate binding affinity calculations within their protein design and optimization process.

Project description:Changes in free energy provide valuable information for molecular recognition, including both ligand-receptor binding thermodynamics and kinetics. Umbrella sampling (US), a widely used free energy calculation method, has long been used to explore the dissociation process of ligand-receptor systems and compute binding free energy. In existing publications, the binding free energy computed from the potential of mean force (PMF) with US simulation mostly yielded "ball park" values with experimental data. However, the computed PMF values are highly influenced by factors such as initial conformations and/or trajectories provided, the reaction coordinate, and sampling of conformational space in each US window. These critical factors have rarely been carefully studied. Here we used US to study the guest aspirin and 1-butanol dissociation processes of β-cyclodextrin (β-CD) and an inhibitor SB2 dissociation from a p38α mitogen-activated protein kinase (MAPK) complex. For β-CD, we used three different β-CD conformations to generate the dissociation path with US windows. For p38α, we generated the dissociation pathway by using accelerated molecular dynamics followed by conformational relaxing with short conventional MD, steered MD, and manual pulling. We found that, even for small β-CD complexes, different β-CD conformations altered the height of the PMF, but the pattern of PMF was not affected if the MD sampling in each US window was well-converged. Because changing the macrocyclic ring conformation needs to rotate dihedral angles in the ring, a bound ligand largely restrains the motion of cyclodextrin. Therefore, once a guest is in the binding site, cyclodextrin cannot freely change its initial conformation, resulting in different absolute heights of the PMF, which cannot be overcome by running excessively long MD simulations for each US window. Moreover, if the US simulations were not converged, the important barrier and minimum were missed. For ligand-protein systems, our studies also suggest that the dissociation trajectories modeled by an enhanced sampling method must maintain a natural molecular movement to avoid biased PMF plots when using US simulations.

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Design of nanobody-based bispecific constructs by in silico affinity maturation and umbrella sampling simulations.

Publications

Design of nanobody-based bispecific constructs by <i>in silico</i> affinity maturation and umbrella sampling simulations.

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