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Proteins achieve their complex functions, such as molecular recognition with high affinity and specificity, through intricate threedimensional geometries in functional sites. To engineer new protein functions, accurate positioning of amino acid functional groups is therefore critical but has remained difficult to achieve by computational methods because of current limitations in the design of new conformations with arbitrary user-defined geometries. Here, we introduce two computational methods capable of generating and predicting new local protein geometries: fragment kinematic closure (FKIC) and loophash kinematic closure (LHKIC). FKIC and LHKIC integrate two approaches: robotics-inspired kinematics of protein conformations and insertion of peptide fragments. We show that FKIC and LHKIC predict native-like conformations at atomic accuracy and with up to 140-fold improvements in sampling efficiency over previous approaches. We then use these methods to create a design protocol, pull into place (PIP), to position functionally important side chains via design of backbone conformations. We validate PIP by remodeling a sizeable active site region in an enzyme and confirming the engineered new conformations of two designs with crystal structures. The described methods can be applied broadly to the design of userdefined geometries for new protein functions. [ABSTRACT FROM AUTHOR]

Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the β- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R-stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+ reductase (FNR)-ferredoxin pair is used as the redox partner. Details of the pro-3R stereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the β-ring-specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis. [ABSTRACT FROM AUTHOR]