8EYZ

Engineered glutamine binding protein bound to GLN and a cobaloxime ligand

Summary for 8EYZ

• Entry DOI: 10.2210/pdb8eyz/pdb
• Descriptor: Amino acid ABC transporter substrate-binding protein, AZIDOBIS (DIMETHYLGLYOXIMATO) PYRIDINECOBALT, SULFATE ION, ... (6 entities in total)
• Functional Keywords: artificial metalloprotein, glutamine-binding protein, conformationally switchable artificial metallorprotein, cobaloxime ligand, metal binding protein
• Biological source: Escherichia coli
• Total number of polymer chains: 12
• Total formula weight: 311114.35

Authors

Bridwell-Rabb, J.,Boggs, D.G.,Olshansky, L.,Fatima, S.,Thompson, P. (deposition date: 2022-10-29, release date: 2022-11-23, Last modification date: 2024-02-14)

Primary citation

Fatima, S.,Boggs, D.G.,Ali, N.,Thompson, P.J.,Thielges, M.C.,Bridwell-Rabb, J.,Olshansky, L.
Engineering a Conformationally Switchable Artificial Metalloprotein.
J.Am.Chem.Soc., 144:21606-21616, 2022

PubMed Abstract: Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)(X) cofactor (dmgH = dimethylglyoxime and X = N, HC, and Pr). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)(CH) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.

PubMed: 36378237
DOI: 10.1021/jacs.2c08885

Experimental method

X-RAY DIFFRACTION (2.99 Å)