Intra-residue methyl–methyl correlations for valine and leucine residues in large proteins from a 3D-HMBC-HMQC experiment

Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a ‘divide and conquer’ approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.

Dual allosteric activation mechanisms in monomeric human glucokinase

Cooperativity in human glucokinase (GCK), the body’s primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme’s small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the 1H-13C heteronuclear multiple quantum coherence (HMQC) spectrum of 13C-Ile–labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the 1H-13C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.