Abstract
The bottom-up design and construction of functional metalloproteins remains a formidable task in biomolecular design. Although numerous strategies have been used to create new metalloproteins, pre-existing knowledge of the tertiary and quaternary protein structure is often required to generate suitable platforms for robust metal coordination and activity. Here we report an alternative and easily implemented approach (metal active sites by covalent tethering or MASCoT) in which folded protein building blocks are linked by a single disulfide bond to create diverse metal coordination environments within evolutionarily naive protein–protein interfaces. Metalloproteins generated using this strategy uniformly bind a wide array of first-row transition metal ions (MnII, FeII, CoII, NiII, CuII, ZnII and vanadyl) with physiologically relevant thermodynamic affinities (dissociation constants ranging from 700 nM for MnII to 50 fM for CuII). MASCoT readily affords coordinatively unsaturated metal centres—including a penta-His-coordinated non-haem Fe site—and well-defined binding pockets that can accommodate modifications and enable coordination of exogenous ligands such as nitric oxide to the interfacial metal centre.
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Data availability
The principal data supporting the findings of this work are available within the figures and the Supplementary Information. Coordinates and structure factor files for Co–H3 (6DYI), apo–CH3 (6DYB), Co–CH3 (6DYC), Fe–CH3 (6DYE), Cu–CH3 (6DYD), Fe–CH3Y (6DYG), Cu–CH3Y (6DYF), VIVO–CH3Y (6DYH), Mn–CH2E (6DY6), Fe–CH2E (6DY4), Mn–CH2EY (6DY8), Fe–CH3Y* (6DYJ), FeNO–CH3Y* (6DYK), and VIVO–CH3Y* (6DYL) have been deposited to the Protein Data Bank with the corresponding PDB ID codes. Additional data that support the findings of this study are available from the corresponding author on request.
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Acknowledgements
This work was supported by the National Science Foundation (grant no. CHE1607145 to F.A.T.). J.R. was supported by a postdoctoral fellowship from the National Institute of General Medical Sciences of the National Institute of Health (grant no. F32GM120981). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource at the Stanford Linear Accelerator Center and the Advanced Light Source at the Lawrence Berkeley National Laboratory, which are supported by the DOE, Office of Science, and Office of Basic Energy Sciences under contracts DE-AC02-76SF00515 and DE-AC02-05CH11231, respectively. We thank C. Moore and M. Gembicky for assistance with XRD experiments.
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J.R. co-conceived the project, designed and performed experiments, analysed data and co-wrote the paper. M.J.F. and M.T.G. performed EPR and Mössbauer experiments. F.A.T. conceived and directed the project and wrote the paper. All authors discussed the results and commented on the manuscript.
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The Supplementary Information File that accompanies this manuscript includes detailed experimental protocols and a description of the materials and methods employed. Supplementary tables and figures include data of interest to specialists and that support the arguments found in the main text.
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Rittle, J., Field, M.J., Green, M.T. et al. An efficient, step-economical strategy for the design of functional metalloproteins. Nat. Chem. 11, 434–441 (2019). https://doi.org/10.1038/s41557-019-0218-9
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DOI: https://doi.org/10.1038/s41557-019-0218-9
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