In Vitro and In Silico Explorations of the Protein Conformational Changes of Corynebacterium glutamicum MshA, a Model Retaining GT-B Glycosyltransferase.

MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded "opened" and liganded "closed" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo "opened" and an artificial generated apo "closed" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-myo-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.

HDX-MS Reveals Substrate-Dependent, Localized EX1 Conformational Dynamics in the Retaining GT-B Glycosyltransferase, MshA.

Glycosyltransferases (GTs) are well-characterized with respect to static 3D structures and molecular dynamics simulations, but there is a lack of reports on in-solution dynamics on time scales relevant to turnover. Here, backbone amide hydrogen/deuterium exchange followed by mass spectrometry (HDX-MS) was used to investigate the in-solution dynamics of the model retaining GT MshA from Corynebacterium glutamicum (CgMshA). CgMshA has a GT-B fold and catalyzes the transfer of N-acetyl-glucosamine (GlcNAc) from UDP-GlcNAc to l-myo-inositol-1-phosphate in the first step in mycothiol biosynthesis. HDX-MS results identify several key regions of conformational changes in response to UDP-GlcNAc binding, including residues 159-198 in the N-terminal domain and residues 323-354 in the C-terminal domain. These regions also exhibited substrate-dependent EX1 exchange kinetics consistent with conformational tension on the milliseconds to seconds time scale. A potential source of this conformational change is the flexible ß4/α5 loop in the C-terminal domain, which sits at the interface of the two domains and likely interacts with the GlcNAc ring of UDP-GlcNAc. In contrast to UDP-GlcNAc, the UDP-CgMshA product complex exhibited severe decreases in deuterium incorporation, suggesting a less dynamic conformation. The HDX-MS results are complemented by solvent viscosity effects of 1.8-2.3 on the CgMshA kcat value, which are consistent with product release as a rate-determining step and possibly a direct role for protein dynamics in catalysis. The identification of in-solution dynamics that are sensitive to substrate binding allows for the proposal of a more detailed mechanism in CgMshA including conformation tension between the donor sugar and the flexible C-terminal domain ß4/α5 loop providing sufficient conformational sampling for substrate-assisted catalysis to occur.

Physical characteristics of 68Ga DOTATATE PET/CT affecting small lesion detectability.

BACKGROUND AND PURPOSE: 68Ga DOTATATE PET/CT protocols are similar to 18F FDG protocols despite differences in physical properties, biodistribution, and tumor uptake. The purpose of this study is to evaluate the impact of scan time (counts), and target activity on signal-to-noise ratio (SNR) in various sized targets, or lesions. To evaluate this, phantom experiments and analysis of clinical 68Ga DOTATATE PET/CT studies were performed. MATERIALS AND METHODS: 68Ga was first compared to 18F in phantom studies to evaluate recovery coefficients and SNR. 68Ga phantom studies were also acquired in list mode, and at varying target activities to evaluate the effects of acquisition time and high target concentrations on SNR in clinically relevant small (8 mm) and larger targets (≥ 12 mm). Clinical studies (n = 50) were analyzed to determine if phantom target concentrations and SNR are present in clinical 68Ga DOTATATE studies at similarly very high tumor activity concentrations (n = 159). RESULTS: In phantoms, recovery coefficient and SUVmax for 68Ga were ~87% of 18F. SNR for 68Ga was ~65% of 18F. For the 68Ga small target (8 mm) at standard T/B = 2.4, increasing scan time from 5 to 15 minutes increased SNR from < 1 to 1.6, and did not result in target identification. Increasing T/B from 2.4 to 10.9, however, dramatically increased SNR from < 1 to 22.3. Increased T/B resulted in clear visibility of the 8 mm target, even for 1-minute scans. In patients, high hepatic tumor SUVmax (27.3±29.6), resulted in high SNR (12.5±9.8). For extrahepatic tumors, high SUVmax (41.6±42.8), resulted in high SNR (43.8±49.9). CONCLUSION: Very high target or T/B, even in small targets, can offset the physical limitations of 68Ga. High target uptake and high T/B are primary factors influencing small lesion detectability.

GABAA Receptor Availability Changes Underlie Symptoms in Isolated Cervical Dystonia.

GABAA receptor availability changes within sensorimotor regions have been reported in some isolated forms of dystonia. Whether similar abnormalities underlie symptoms in cervical dystonia is not known. In the present study, a total of 15 cervical dystonia patients and 15 age- and sex-matched controls underwent 11C-flumazenil PET/CT scanning. The density of available GABAA receptors was estimated using a Simplified Reference Tissue Model 2. Group differences were evaluated using a two-sample T-test, and correlations with dystonia severity, as measured by the Toronto Western Spasmodic Torticollis Rating Scale, and disease duration were evaluated using a regression analysis. Voxel-based analyses revealed increased GABAA availability within the right precentral gyrus in brain motor regions previously associated with head turning and the left parahippocampal gyrus. GABAA availability within the bilateral cerebellum was negatively correlated with dystonia severity, and GABAA availability within the right thalamus and a variety of cerebellar and cortical regions were negatively correlated with disease duration. While GABAA availability changes within primary motor areas could represent a partial compensatory response to loss of inhibition within sensorimotor network, GABAergic signaling impairment within the cerebellum may be a key contributor to dystonia severity.

Parathyroid imaging: the importance of pinhole collimation with both single- and dual-tracer acquisition.

UNLABELLED: Our objective was to rigorously compare pinhole and parallel-hole collimation in an intrapatient, intrastudy design in 2 parathyroid imaging protocols: the first was dual-phase (99m)Tc-sestamibi imaging, and the second was dual-phase (99m)Tc-sestamibi plus dual-tracer ((99m)Tc-sestamibi and (123)I) simultaneous-acquisition subtraction imaging. METHODS: Thirty-three patients with 37 surgically proven nonectopic parathyroid adenomas were evaluated. Anterior pinhole and parallel-hole images of the neck were available for (99m)Tc-sestamibi at 15 min and 3 h, and for simultaneously acquired (99m)Tc-sestamibi and (123)I subtraction at 15 min, all from a single study. The images were modified so that all had a square border and so that the thyroid filled approximately three quarters of the image. The images were evaluated by 2 experienced nuclear medicine physicians who did not know the surgical results or whether the images were acquired with pinhole or parallel-hole collimation. The observers indicated the location of any identified adenoma and graded the certainty of diagnosis on a 3-point scale. RESULTS: The localization success rate for the 2 observers combined for the single-tracer dual-phase images was 66.2% with pinhole collimation and 43.2% with parallel-hole collimation (P < 0.0001). The localization success rate with the addition of the dual-tracer simultaneous-acquisition subtraction image was 83.8% with pinhole collimation and 62.2% with parallel-hole collimation (P = 0.0018). In addition, the degree of certainty of localization was greater with pinhole collimation with both imaging protocols (P < 0.001 in both cases). CONCLUSION: In the anterior projection, pinhole collimation is superior to parallel-hole collimation for parathyroid imaging with either dual-phase (99m)Tc-sestamibi or dual-phase (99m)Tc-sestamibi plus dual-tracer ((99m)Tc-sestamibi and (123)I) simultaneous-acquisition subtraction.