PROJECT SUMMARY
Protein-ligand binding events underlay all life processes. Protein design tests and extends our knowledge of
protein folding and function through the creation of proteins from scratch. This proposal aims to develop a
computational method for the design of proteins that bind to any small molecule with high affinity and
selectivity. The state-of-the-art in ligand-binding protein design critically relies on random experimental
optimization and screening. If we truly understand how proteins bind small molecules, we should be able to go
directly from computer models to tight binders. The hypothesis that drives this proposal is that proteins use a
vast but now enumerable number of molecular interaction motifs combinatorially throughout evolution to create
the binding sites of modern-day proteins. Computational methods will be employed to uncover this set of
interactions in the large database of protein structures available in the protein databank (PDB). Binding sites
will be designed by sampling motifs for all functional groups of a ligand onto a protein backbone. We call this
design method Convergent Motifs for Binding Sites (COMBS). COMBS was used to design ABLER, the first
ligand-binding protein designed from scratch to bind its target ligand—the antithrombotic drug apixaban—with
an unprecedentedly high affinity, without experimental optimization of sequence. ABLER has potential clinical
relevance as an anti-clotting antidote, although that is outside the scope of the proposal. High-resolution crystal
structures of ABLER agree with the design model, both in overall topology and the intended molecular
interactions with the ligand. Aim 1 of this proposal focuses on designing variants of ABLER to increase affinity
and probe the molecular bases for the observed drug-protein interactions. Aim 2 focuses on the role of water in
ligand-binding protein design, motivated by the water-mediated protein-ligand interaction found in the crystal
structure. In this Aim, I will curate a database of water-protein interactions from the PDB and use these to
sample water-mediated protein-ligand interactions during design. I will also learn to use explicit-water
molecular dynamics simulations to critically assess the roles of water in binding. Aim 3 uses COMBS to redesign
the binding site of pyrrolysine tRNA synthetase for incorporation of charged unnatural amino acids (such as
sulfotyrosine) into mammalian cells, since laboratory evolution and library screens for this goal have so far been
unsuccessful. These aims will augment my training in molecular biology, computational protein design, and
protein structural characterization (X-ray crystallography and NMR). The K99 portion of this work in the
DeGrado lab will expose me to all aspects of the scientific process, from inception to publication. Bill is a world
expert in protein design, and his insight is critical to the success of the project. At UCSF, I will gain much through
my regularly scheduled meetings with Ethan Weiss, who brings the perspective of a physician scientist with a
long history of antithrombotic research and clinical applications. My collaboration with UCSF professor Lei
Wang will expose me to the field of unnatural amino acid incorporation and will be critical for applying COMBS
to the most impactful targets for mimics of post-translational modifications. The research and training proposed
herein will greatly complement my current skillset and background, which will be essential to my research
program as I transition into an independent principal investigator.