Automated method of computational enzyme identification and design
Inventors
Zanghellini, Alexandre • Ban, Yih-En Andrew • ALTHOFF, Eric Anthony • Grabs, Daniela • Azoitei, Mihai Luchian
Assignees
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Publication Number
US-10025900-B2
Publication Date
2018-07-17
Expiration Date
Abstract
The invention provides computational methods for engineering, selecting, and/or identifying proteins with a desired activity. Further provided are automated computational design and screening methods to engineer proteins with desired functional activities including, but not limited to ligand binding, catalytic activity, substrate specificity, regioselectivity and/or stereoselectivity.
Core Innovation
The invention describes an automated computational pipeline for making a protein having enzymatic activity by combining template-derived functional-site constraints with homology-based candidate selection, structural modeling, constrained ligand docking, and optimization of amino-acid positioning. A template protein having the enzymatic activity is selected from carbonyl reductase, decarboxylase, and beta-lactamase, and a template structure is obtained to serve as the basis for subsequent design steps.
A functional site description is prepared based on the template structure and includes amino acid identities for each functional site amino acid residue, rotameric states for each functional residue, rotameric states for each ligand, and geometric placement of the functional residues with respect to the ligands. Candidate amino-acid sequences are computationally selected having structural homology and/or sequence homology to the template protein, and structural models are provided for each selected sequence.
For each modeled sequence, amino-acid sequences satisfying the functional site description are selected by computationally docking a ligand followed by optimizing positioning of amino-acid side chains and main chain atoms. At least one sequence that satisfies the functional site description is recombinantly expressed and its enzymatic activity is confirmed, thereby making the protein having the enzymatic activity.
Claims Coverage
The independent claim covers a method pipeline with six core inventive elements: template acquisition, functional-site description preparation with rotameric and geometric constraints, homology-based sequence selection, structural modeling, constrained ligand docking with side-chain and main-chain optimization, and recombinant expression with enzymatic activity confirmation.
Template selection for enzymatic activity proteins
Obtaining a template structure of a template protein having the enzymatic activity, wherein the template protein is selected from the group consisting of: a carbonyl reductase, decarboxylase, and beta-lactamase.
Functional site description from template structure with rotameric and geometric constraints
Preparing a functional site description based on the template structure, wherein the functional site description comprises amino acid identities for each functional site amino acid residue, rotameric states for each functional residue, rotameric states for each ligand, and geometric placement of the functional residues with respect to said ligands.
Homology-based computational selection of candidate sequences
Computationally selecting one or more amino acid sequences having structural homology and/or sequence homology to the template protein having the enzymatic activity.
Structural modeling for each selected amino-acid sequence
Providing a structural model for each of the amino acid sequences selected in step (c).
Constrained ligand docking with side-chain and main-chain optimization
Selecting the amino acid sequences satisfying the functional site description comprising steps of computationally docking a ligand and optimizing positioning of amino acid side chains and main chain atoms of the amino acid sequences.
Recombinant expression and enzymatic activity confirmation
Recombinantly expressing and confirming the enzymatic activity for at least one of the amino acid sequences that satisfies the functional site description selected from step (e), thereby making the protein having the enzymatic activity.
Overall claim coverage is centered on a template-to-functional-site-description-to-homology-candidate selection-to-structural modeling-to-constrained docking and optimization workflow, ending with recombinant expression and confirmation of enzymatic activity for at least one selected sequence.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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