Order-Disorder Transitions Govern Kinetic Cooperativity and Allostery of Monomeric Human Glucokinase

Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity-onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder-order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder-order cycle of the small domain as a "time-delay loop," which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.NIH [1R01DK081358]NIHNSF [MCB-0918362]NSFAmerican Heart AssociationAmerican Heart Associatio

Targeting the Sphingolipid Rheostat in Gliomas

Gliomas are highly aggressive cancer types that are in urgent need of novel drugs and targeted therapies. Treatment protocols have not improved in over a decade, and glioma patient survival remains among the worst of all cancer types. As a result, cancer metabolism research has served as an innovative approach to identifying novel glioma targets and improving our understanding of brain tumors. Recent research has uncovered a unique metabolic vulnerability in the sphingolipid pathways of gliomas that possess the IDH1 mutation. Sphingolipids are a family of lipid signaling molecules that play a variety of second messenger functions in cellular regulation. The two primary metabolites, sphingosine-1-phosphate (S1P) and ceramide, maintain a rheostat balance and play opposing roles in cell survival and proliferation. Altering the rheostat such that the pro-apoptotic signaling of the ceramides outweighs the pro-survival S1P signaling in glioma cells diminishes the hallmarks of cancer and enhances tumor cell death. Throughout this review, we discuss the sphingolipid pathway and identify the enzymes that can be most effectively targeted to alter the sphingolipid rheostat and enhance apoptosis in gliomas. We discuss each pathway’s steps based on their site of occurrence in the organelles and postulate novel targets that can effectively exploit this vulnerability

The patatin-like protein PlpD forms structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane

Abstract Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded β-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal β-barrel domain. Challenging the current dogma, we find that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring β-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions

Isoleucine side chain solution NMR spectra along the GCK reaction coordinate.

<p>Crystal structures of GCK in (A) unliganded (PDB 1V4T), (B) glucose-bound (PDB 3IDH), and (C) glucose and AMP-PNP-bound forms (PDB 3FGU) depicting the location of isotopically labeled side chains used in this study. Ile Cα positions and tryptophan Cα side chains are depicted as red and yellow spheres, respectively. The large and the small domains are represented in gray and blue, respectively. β-hairpin/loop 151–179 is colored in magenta, glucose is in green, and AMP–PNP in orange. 2D ¹H-¹³C HMQC NMR spectra of Cδ1 methyl groups of (D) unliganded, (E) glucose-bound, and (F) glucose and AMP–PNP-bound forms of GCK. Assignments of Ile residues in the large domain are in gray with label “L,” Ile residues in the small domain are in blue with label “S,” and Ile residues in the 151–179 loop are in magenta. The average Ile Cδ1 chemical shift of intrinsically disordered Ile side chains deposited in the BMRB database is displayed as an open red circle in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001452#pbio-1001452-g001" target="_blank">Figure 1D</a> (for more details, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001452#pbio.1001452.s004" target="_blank">Figure S4</a>).</p

Structural, dynamic, and kinetic descriptions of GCK activating states.

<p>(A) Crystal structure of the GCK–activator complex depicting the spatial vicinity of the regions involved in allosteric communication. The 151–179 β-hairpin is shown in magenta, the α13-helix in blue, and the Cαs of Ile-159, Ile-163, and Trp-167 residues are colored in red (Ile) and yellow (Trp) spheres. Glucose is colored in green, and the synthetic allosteric activator in cyan. The side chain of Lys-169, which is represented by magenta sticks, is hydrogen-bonded to O6 of glucose. Val-452 and Ile-159 side chains are located within 5 Å of each other and of the activator thiazole ring. (B) Kinetic response of the α13-helix variant (green), and wild-type GCK in the absence (blue) and presence (red) of saturating activator (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001452#pbio.1001452.s010" target="_blank">Table S1</a>). 2D ¹H-¹³C HMQC spectra of (C) unliganded GCK, (D) activator-bound complex, and (E) unliganded α13-helix variant, specifically labeled with Ile ¹³C(δ1) methyl groups. 2D ¹H-¹⁵N HSQC spectra of (F) unliganded GCK, (G) glucose-bound complex, and (H) unliganded α13-helix variant, with Trp-¹⁵Nε cross-peaks assigned by black labels. The yellow box highlights the position of the W167 cross-peak. The asterisk indicates that W167 is the main contributor to the intensity of the unliganded cross-peak (for more details, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001452#pbio.1001452.s003" target="_blank">Figure S3</a>).</p

Mechanism of GCK kinetic cooperativity.

<p>(A) The small domain of unliganded GCK is intrinsically disordered, giving rise to a broad conformational ensemble. (B) Glucose binding, activator binding, or an activating PHHI-associated mutation promotes folding of the disordered regions in the small domain, narrowing the conformational distribution. Upon formation of the GCK–glucose binary complex, ATP binds and catalysis proceeds with little additional reorganization. (C) Following product release, ordered unliganded GCK persists until the small domain undergoes an order–disorder transition on the millisecond time scale, allowing access to the “time delay loop” (red): Under low glucose concentrations, the delay loop is operational, leading to slow turnover and kinetic cooperativity. Under high glucose concentrations (or when GCK is activated), the delay loop is effectively bypassed, turnover is fast, and cooperativity is eliminated (green).</p