relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data

Morin, Sébastien; Linnet, Troels Emtekær; Lescanne, Mathilde; Schanda, Paul; Thompson, Gary S.; Tollinger, Martin; Teilum, Kaare; Gagné, Stéphane M.; Marion, Dominique; Griesinger, Christian; Blackledge, Martin; d’Auvergne, Edward J.

doi:10.1093/bioinformatics/btu166

Cited by 52 publications

(35 citation statements)

References 22 publications

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“…Relaxation dispersion curves obtained at two static magnetic fields were generated by plotting R 2 vs. 1/τ cp using GraphPad Prism 7.0 (GraphPad Software) and fit simultaneously using the fast-limit CPMG equation, where uncertainty values were obtained from replicate spectra. Dual field relaxation dispersion data were also processed with RELAX (41-43) using the R2eff, NoRex, and MMQCR72 (two-site Carver-Richards) models (44). Relax was used to globally fit residues undergoing dispersion in V12A, K19A, V48A, and D98A IGPS, and statistical analyses of independent and global models indicated that only V12A IGPS was well-described by a global dynamic process based on the Akaike information criterion (28,45,46).…”

Section: Methodsmentioning

confidence: 99%

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

Lisi

East

Batista

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, meaning that its catalytic rate is critically dependent on activation by its allosteric ligand, N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). The allosteric mechanism of IGPS is reliant on millisecond conformational motions for efficient catalysis. We engineered four mutants of IGPS designed to disrupt millisecond motions and allosteric coupling to identify regions that are critical to IGPS function. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments and NMR chemical shift titrations reveal diminished enzyme flexibility and a reshaping of the allosteric connectivity in each mutant construct, respectively. The functional relevance of the observed motional quenching is confirmed by significant reductions in glutaminase kinetic activity and allosteric ligand binding affinity. This work presents relevant conclusions toward the control of protein allostery and design of unique allosteric sites for potential enzyme inhibitors with regulatory or therapeutic benefit.allostery | NMR | community networks | millisecond motions T he underlying principles of allosteric regulation have been a focal point of enzymology and structural biology for decades as studies of allostery have evolved from two phenomenological models (1, 2) to recognize structural and conformational ensembles that define a broad range of enzymatic states (3-6). As a result, a great deal of emphasis has been placed on understanding dynamic contributions to allostery (7-12) and factors that enable small fluctuations in local conformations of enzymes to propagate information over large distances. A well-developed understanding of dynamic allostery opens up numerous avenues for insight into protein engineering (13,14), drug design (15), and mechanistic biochemistry (16)(17)(18)(19)(20)(21)(22). To establish universal principles for relevant enzymes, however, a link between dynamics and function must be made.The heterodimeric enzyme imidazole glycerol phosphate synthase (IGPS) plays a crucial role in amino acid and purine biosynthesis in bacteria and other microorganisms by sequentially catalyzing the hydrolysis of glutamine (Gln) in its HisH subunit and the cyclization of the substrate and allosteric effector N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) in its HisF subunit (Scheme 1). IGPS is a V-type allosteric enzyme that is inactive unless PRFAR is bound, and communication between the active and effector sites is driven by the enhancement of conformational flexibility, namely, fluctuations taking place on the millisecond timescale (22-24). Based on NMR and computational evidence, these motions are believed to propagate from the PRFAR binding site to the glutaminase binding site, thereby disrupting a hydrogen bond between V51 and P10 of HisH. This H bond prevents formation of the oxyanion hole site necessary for glutamine hydrolysis, and its disruption allows IG...

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Section: Methodsmentioning

confidence: 99%

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

Lisi

East

Batista

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Relaxation dispersion curves were generated by plotting R 2 vs. 1/ τ cp using RELAX(Bieri et al, 2011; d’Auvergne et al, 2008a, b) with the R2eff, NoR ex , and MMQCR72 (full two-site Carver-Richards) models. (Morin et al, 2014) Relaxation dispersion data obtained at two static magnetic fields were fit simultaneously using the fast CPMG Equation and uncertainty values were obtained within RELAX from replicate spectra.…”

Section: Experimental Nmr Proceduresmentioning

confidence: 99%

Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators

et al. 2016

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1. Summary The allosteric mechanism of the heterodimeric enzyme imidazole glycerol phosphate synthase was studied in detail with solution NMR spectroscopy and molecular dynamics simulations. We studied IGPS in complex with a series of allosteric activators corresponding to a large range of catalytic rate enhancements (26 – 4900 fold), in which ligand binding is entropically driven. Conformational flexibility on the millisecond timescale plays a crucial role in intersubunit communication. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments probing Ile, Leu, and Val methyl groups reveal that the apo- and glutamine-mimicked complexes are static on the millisecond timescale. Domain-wide motions are stimulated in the presence of the allosteric activators. These studies, in conjunction with ligand titrations, demonstrate that the allosteric network is widely dispersed and varies with the identity of the effector. Further, we find that stronger allosteric ligands create more conformational flexibility on the millisecond timescale throughout HisF. This domain-wide loosening leads to maximum catalytic activity.

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“…However, the REC residues, such as Ile 3 , Gly 24 , Val 26 , Cys 27 , Asp 28 , Ser 46 , Ile 127 and Val 136 , which are located in the REC-DBD interface, exhibit only one set of NMR resonances in the DNA-bound state (Fig. 4a-c).…”

Section: Resultsmentioning

confidence: 99%

“…11). Measurements at both 600 and 850 MHz were fitted individually by use of the software relax 28 to a two-site exchange process (A2B), yielding populations (p A and p B ), exchange rate constants (k ex ¼ k AB þ k BA ) and the 1 H and 13 C chemical shift differences between the two states 27 (Supplementary Table 4). In total, 12 PmrA methyl groups exhibit slow dynamics on binding to DNA.…”

Section: Resultsmentioning

confidence: 99%

“…For relaxation dispersion, the effective decay rate (R 2,eff ) was calculated as R 2,eff ¼ ( À 1/T) ln[I(n CPMG )/I(0)], where I(n CPMG ) and I(0) are the intensities of peaks recorded with and without the CPMG intervals, respectively. CPMG relaxation dispersion measurements at both 600 and 850 MHz were fitted individually by the software relax 28 to a two-site exchange process. The detailed auto-analysis process can be found in the online manual for relax (http://www.nmr-relax.com/docs.html).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

Hsiao¹

2016

Acta Cryst Sect A

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relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data

Cited by 52 publications

References 22 publications

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

Dissecting Dynamic Allosteric Pathways Using Chemically Related Small-Molecule Activators

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

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