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Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin.

ABSTRACT: Rational protein design has been proven to be a powerful tool for creating functional artificial proteins. Although many artificial metalloproteins with a single active site have been successfully created, those with dual active sites in a single protein scaffold are still relatively rare. In this study, we rationally designed dual active sites in a single heme protein scaffold, myoglobin (Mb), by retaining the native heme site and creating a copper-binding site remotely through a single mutation of Arg118 to His or Met. Isothermal titration calorimetry (ITC) and electron paramagnetic resonance (EPR) studies confirmed that a copper-binding site of [3-His] or [2-His-1-Met] motif was successfully created in the single mutant of R118H Mb and R118M Mb, respectively. UV/Vis kinetic spectroscopy and EPR studies further revealed that both the heme site and the designed copper site exhibited nitrite reductase activity. This study presents a new example for rational protein design with multiple active sites in a single protein scaffold, which also lays the groundwork for further investigation of the structure and function relationship of heme/non-heme proteins.

SUBMITTER: Shu XG

PROVIDER: S-EPMC5125789 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin.

Shu Xiao-Gang XG Su Ji-Hu JH Du Ke-Jie KJ You Yong Y Gao Shu-Qin SQ Wen Ge-Bo GB Tan Xiangshi X Lin Ying-Wu YW

ChemistryOpen 20160308 3

Rational protein design has been proven to be a powerful tool for creating functional artificial proteins. Although many artificial metalloproteins with a single active site have been successfully created, those with dual active sites in a single protein scaffold are still relatively rare. In this study, we rationally designed dual active sites in a single heme protein scaffold, myoglobin (Mb), by retaining the native heme site and creating a copper-binding site remotely through a single mutatio ...[more]

PMID: 27933225

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Project description:ObjectiveBiologic drugs, such as the anti-tumor necrosis factor (anti-TNF) antibody adalimumab, have represented a breakthrough in the treatment of rheumatoid arthritis. Yet, concerns remain over their lack of efficacy in a sizable proportion of patients and their potential for systemic side effects such as infection. Improved biologic prodrugs specifically targeted to the site of inflammation have the potential to alleviate current concerns surrounding biologic anticytokine therapies. The purpose of this study was to design, construct, and evaluate in vitro and ex vivo the targeting and antiinflammatory capacity of activatable bispecific antibodies.MethodsActivatable dual variable domain (aDVD) antibodies were designed and constructed to target intercellular adhesion molecule 1 (ICAM-1), which is up-regulated at sites of inflammation, and anti-TNF antibodies (adalimumab and infliximab). These bispecific molecules included an external arm that targets ICAM-1 and an internal arm that comprises the therapeutic domain of an anti-TNF antibody. Both arms were linked to matrix metalloproteinase (MMP)-cleavable linkers. The constructs were tested for their ability to bind and neutralize both in vitro and ex vivo targets.ResultsIntact aDVD constructs demonstrated significantly reduced binding and anti-TNF activity in the prodrug formulation as compared to the parent antibodies. Human synovial fluid and physiologic concentrations of MMP enzyme were capable of cleaving the external domain of the antibody, revealing a fully active molecule. Activated antibodies retained the same binding and anti-TNF inhibitory capacities as the parent molecules.ConclusionThe design of a biologic prodrug with enhanced specificity for sites of inflammation (synovium) and reduced specificity for off-target TNF is described. This construct has the potential to form a platform technology that is capable of enhancing the therapeutic index of drugs for the treatment of RA and other inflammatory diseases.

Dataset Information

Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin.

Publications

Rational Design of Dual Active Sites in a Single Protein Scaffold: A Case Study of Heme Protein in Myoglobin.

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