# Exploring Trop-2–Nanobody PPI Interactions through Molecular Dynamics Simulations and Biovalidations

## TL;DR

This study uses simulations and experiments to understand how nanobodies bind to Trop-2, a cancer-related protein, to improve targeted cancer therapies.

## Contribution

The study identifies key residues and binding mechanisms of Trop-2–nanobody interactions and demonstrates how CDR3 redesign affects binding affinity.

## Key findings

- MD simulations confirmed the most reliable binding pose between Trop-2 and nanobodies.
- Key residues in the C-terminal domain and α-helix region of Trop-2 were identified as critical for binding.
- Redesigned CDR3 variants showed significant differences in binding affinity, with Nb-7 having the highest.

## Abstract

The transmembrane
glycoprotein Trop-2 has garnered significant
attention as a potential therapeutic target due to its involvement
in various malignancies, including breast, lung, and prostate cancers.
Specifically, the exploration of Trop-2-specific antibodies and nanobodies
has emerged as a promising avenue for innovative treatment strategies.
Despite advancements, a comprehensive understanding of the intricate
molecular interactions between Trop-2 and specific nanobodies remains
elusive. In this study, molecular dynamics (MD) simulations with MM/GBSA
were employed to investigate the binding poses, key residues, and
detailed interactions between Trop-2 and three distinct nanobodies
(Nb60, Nb65, and Nb108), which will further guide the development
of Trop-2-targeting nanobodies. Our findings corroborated the docking
results, highlighting the reliability of binding pose 1. Additionally,
our simulations elucidated key residues involved in the interaction
interface, particularly within the C-terminal cysteine-poor domain
(CPD) and an α-helix region (S170–Y185) on Trop-2. Furthermore,
we identified the critical role of the complementarity-determining
region 3 (CDR3) length and the key residues involved in the binding
of nanobodies to Trop-2. Potential key residues predicted by MD simulations
guided the redesign of the CDR3 region of Nb60, generating variants
Nb-6, Nb-7, and Nb-14. Experimental data showed that alterations in
the CDR3 significantly impacted binding affinity, with Nb-7 exhibiting
the highest affinity (K
d value of 7.931
μM), whereas Nb-6 and Nb-14 showed reduced or no binding. This
comprehensive analysis provides valuable insights into the molecular
mechanisms governing Trop-2–nanobody interactions, facilitating
future nanobody engineering efforts for targeted cancer therapy.