A Vast Navigable Protein Sequence Subspace

Abstract

Fitness landscapes govern evolution. Our empirical understanding of their structure has primarily been informed by evolutionary history. Here, we combine high-throughput DNA synthesis and machine learning, without training on evolutionary priors, to build a high-resolution map of the genome packaging landscape of the Adeno-associated Virus capsid protein. We assay 232,123 mutants around Adeno-Associated Virus 2 (AAV2) including 15 neighborhoods centered on seed sequences within 5-21 mutations of the wild type sequence. Local neighborhoods differ sharply in mutational tolerance and epistatic detail, yet much of the quantitative variation is captured by a simple pattern: sitewise effects transformed by global epistasis. Strikingly, using targeted sampling of only a few thousand mutants, we uncover a vast percolating network of function-preserving ridges embedded within a largely non-functional landscape. These ridges support billions of distinct trajectories of up to 667 steps that connect all sampled neighborhoods through functional single mutations. Designed variants with mutations distinct from the evolutionary background retain packaging at distances where randomly generated mutants rarely survive. These include capsids that combine up to 7 mutations that are largely absent from the evolutionary record, and which are each individually lethal in the 15 diverse backgrounds we tested. Our results reveal rich subspace structure resembling landscape orebodies: reservoirs of highly viable and navigable sequence space defined by simple constraints. These subspaces can be discovered through targeted sampling and can open access to vast troves of proteins that may be valuable for downstream applications such as therapeutics.