Computational Design of Soluble CCR8 Analogues with Preserved Antibody Binding

Abstract

G protein-coupled receptors (GPCRs) represent the largest class of drug targets, yet their membrane-embedded nature poses significant challenges for structural studies and therapeutic development. Here, we report the successful computational design and experimental validation of soluble CCR8 analogues that maintain native antibody binding properties. Using an integrated pipeline combining ProteinMPNN-sol sequence design, and structure-based filtering, we generated 13 CCR8 analogues from 272 initial designs across three N-terminal truncation strategies. Experimental validation confirmed 62% success rate (8/13 designs) with protein yields of 1.19-73.72 mg/L in aqueous buffer, representing a significant improvement over traditional membrane protein production method. Surface plasmon resonance analysis demonstrated that all analogues retained mAb1 binding with dissociation constants ranging from 77-857 nM, comparable to wild-type CCR8 (KD = 190 nM). Despite extensive sequence divergence (10-13% identity with wild-type CCR8), structural integrity was preserved as evidenced by binding affinity maintenance and computational structural validation. This work demonstrates the feasibility of computationally designing functional soluble analogues of challenging membrane proteins, with implications for accelerating drug discovery, antibody development, and structural biology studies. Our approach addresses critical limitations in membrane protein accessibility while preserving native epitope presentation, opening new avenues for therapeutic target characterization and binder discovery.