Unraveling a Ligand‐Induced Twist of a Homodimeric Enzyme by Pulsed Electron–Electron Double Resonance

Abstract

Mechanistic insights into protein-ligand interactions can yield chemical tools for modulating protein function and enable their use for therapeutic purposes.For the homodimeric enzyme tRNA-guanine transglycosylase (TGT), ap utative virulence target of shigellosis,l igand binding has been shown by crystallography to transform the functional dimer geometry into an incompetent twisted one.H owever,c rystallographic observation of both end states does neither verify the ligandinduced transformation of one dimer into the other in solution nor does it shed light on the underlying transformation mechanism. We addressed these questions in an approachthat combines site-directed spin labeling (SDSL) with distance measurements based on pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. We observed an equilibrium between the functional and twisted dimer that depends on the type of ligand, with ap yranose-substituted ligand being the most potent one in shifting the equilibrium towardt he twisted dimer.O ur experiments suggest ad issociation-association mechanism for the formation of the twisted dimer upon ligand binding.

Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis‐Cu2+(NTA) Motif

“…The method has been applied to biomolecules in buffer, [10] in membranes, [11] and within cells [12] to investigate conformational changes in proteins and oligonucleotides, [13][14][15][16] to unravel the timescale of conformational changes, [17,18] to localize metal ions, [19][20][21] and to obtain insights into bio-macromolecular dynamics [22] and complex formation. [23] Although bio-macromolecules can contain inherent paramagnetic centers, such as metal ions [24,25] or amino acid radicals, [26] they are in most cases diamagnetic. Therefore, a paramagnetic center is introduced by site-directed spin labeling (SDSL).…”

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase

“…This unproductive quaternary structure, whose formation is triggered by certain inhibitors of this enzyme, is formally generated from the conventional active dimer by a ca. 130° rotation of one subunit against the other one. − However, the similarity of protein packing in the crystal structure of murine QTRT1 with the twisted dimer of bacterial TGT seems to be accidental, and the corresponding protein/protein interface may represent a mere crystal contact. While the protein/protein interface of the bacterial twisted dimer virtually covers the same area as the conventional homodimer interface (>1600 Å 2 ), the contact area between both protein molecules in the asymmetric unit of the murine QTRT1 crystal amounts to less than 800 Å 2 .…”

Modeling of Cu(ii)-based protein spin labels using rotamer libraries

“…Pulsed dipolar EPR spectroscopy coupled with site-directed spin labeling (SDSL) is a powerful tool for obtaining sparse distance restraints [1][2][3][4][5][6][7] that relate to the structure and conformational plasticity of proteins. When a protein is engineered to have two spin labels, distance measurements between the labels provide information about conformational changes, [8][9][10][11][12][13][14][15][16][17][18] ligand binding site location, [19][20][21][22][23][24] and tertiary and quaternary structures of large proteins and protein assemblies. [25][26][27][28][29][30][31][32] In these applications, the prediction of spin label conformations is invaluable.…”