Evolutionary conservation and local structural context of DNAJC14 W576

Introduction: DnaJ heat-shock protein family (Hsp40) member C14 (DNAJC14) is an endoplasmic-reticulum-associated co-chaperone required for regulated non-structural protein 2–3 (NS2–3) processing in classical pestiviruses. Functional studies establish the importance of porcine W576, but its residue-level structural context is unresolved. This study aimed to define the evolutionary conservation and predicted local structural context of DNAJC14 W576. Materials and methods: We combined raw-sequence and phylogeny-weighted conservation analyses with a 30-model AlphaFold/ColabFold ensemble, buried-residue contact screening, voxel-based local-void and repacking analysis, archived multiple-sequence-alignment (MSA) auditing, and FoldX comparison of eight substitutions at W576 and buried-tryptophan controls W492 and W601. Results: The W576-equivalent residue was tryptophan (W) in all 288 site-mappable records from the 300-record dataset and remained invariant under alternative and 10,000 random representative-selection schemes. Phylogeny weighting gave an effective sequence number of 90.8, while 28/71 local positions were also invariant. Model W575 (published W576) was the most buried tryptophan and ranked 3/67 among buried residues for non-adjacent side-chain contacts. W576A left 85.75 ± 1.12 Å 3 (83.3%) of the model-defined local volume unfilled with limited neighbouring-side-chain repacking. Wild-type (WT) versus W576A AlphaFold coordinate differences were sub-ångström and interpreted descriptively because archived WT and mutant MSAs differed. FoldX penalty ordering was partly shared by W492 and W601, while W576 penalties were larger for most substitutions. Conclusions: W576 is invariant across sampled vertebrate DNAJC14 orthologs and occupies a deeply buried, densely contacted predicted environment. Alanine substitution leaves persistent model-defined local free volume, while control sites show that substitution ordering is not W576-specific.