Toward Robust Characterization of Dynamic Binding Pockets: Lessons from the HBV Capsid Assembly Modulator Site

Protein function often depends on ligand binding pockets that fluctuate among conformational states, altering their size, shape, topology, and accessibility, yet quantitative comparison of these dynamic cavities remains challenging because their boundaries are often inherently ambiguous. The measure volinterior algorithm uses fuzzy-boundary detection to characterize enclosed molecular spaces; here, the hepatitis B virus (HBV) capsid assembly modulator (CAM) binding site is used as a model system to develop and validate a practical workflow for applying the method to dynamic protein binding pockets. The resulting methodology provides practical guidance for parameter selection and evaluation, establishes a standardized protocol for quantitative characterization of the HBV CAM pocket, and demonstrates robust, reproducible performance across conformational ensembles derived from molecular dynamics (MD) simulations. More broadly, this work provides a reproducible strategy for adapting measure volinterior to other dynamic binding pockets, enabling consistent comparison of pocket geometry among independent structural studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/743403v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@d460b5org.highwire.dtl.DTLVardef@11915c2org.highwire.dtl.DTLVardef@1e37524org.highwire.dtl.DTLVardef@1fc1b7_HPS_FORMAT_FIGEXP M_FIG C_FIG