HDX-MS guided drug discovery: small molecules and biopharmaceuticals

Marciano, David; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.

doi:10.1016/j.sbi.2014.08.007

Cited by 82 publications

(83 citation statements)

References 82 publications

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“…HDX measures the incorporation of deuterons from heavy water (D 2 O) with mass spectrometry over time to probe the secondary structure. HDX has emerged as a sensitive and rapid technique to identify alterations in conformational dynamics in proteinprotein or protein-ligand interactions (35)(36)(37). The HDX heat map of apo-G␣ i1 (data not shown) agrees closely with previous reports of G protein structure and overall stability (38,39), whereas the HDX heat map of apo-RGS14 indicates that RGS14 is highly dynamic in solution (Fig.…”

Section: And D) Following Activation Of Hela Cells With Alfsupporting

confidence: 82%

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

Brown¹,

Goswami

Branch³

et al. 2015

Journal of Biological Chemistry

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Background: RGS14 binds distinct forms of active and inactive G␣ proteins through its RGS domain and GPR motif. Results: Inactive G␣ i1 -GDP binding of the GPR motif does not preclude RGS action on active G␣ o -GTP. Conclusion: RGS14 simultaneously binds active G␣ o and inactive G␣ i1 while retaining GAP activity. Significance: These findings clarify our understanding of how RGS14 integrates signaling by distinct G protein subunits.

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Section: And D) Following Activation Of Hela Cells With Alfsupporting

confidence: 82%

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

Brown¹,

Goswami

Branch³

et al. 2015

Journal of Biological Chemistry

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“…To identify the c-di-GMP binding site of CuxR, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS), also because we were unsuccessful to obtain crystals of CuxR bound to c-di-GMP. This method allows determining conformational changes within proteins upon their interaction with ligands (45,46). Specifically, CuxR and c-di-GMP-bound CuxR were incubated in deuterated buffer for 0.5, 2, or 10 min.…”

Section: A C-di-gmp-dependent Homodimer Of Cuxr Activates the Uxs1mentioning

confidence: 99%

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

Schäper

Steinchen

Krol

et al. 2017

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

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Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal “RxxxR” motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP–responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP–responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.

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“…By massively increasing its interest in protein therapeutics within these last years, the biopharmaceutical industry became particularly interested in improving and developing spatially resolved analytical techniques like HDX-MS for its needs in both research and quality control. (14,15) Since so far HDX-MS was mostly implemented on monomeric proteins (16)(17)(18)(19)(20)(21)(22) and only a few studies have been conducted on homo-oligomeric proteins (23)(24)(25)(26)(27) or protein complexes, (28)(29)(30)(31) efforts have to be continued for extracting the dynamic determinants of complexes.…”

Section: Introductionmentioning

confidence: 99%

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

2017

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Most oligomeric proteins become active only after assembly, but why oligomerization is required to support function is not well understood. Here, we address this question using the WT and a destabilized mutant (D93N) of the hexameric nucleoside diphosphate kinase from the pathogen Mycobacterium tuberculosis (Mt-NDPK). The conformational dynamics and oligomeric states of each were analyzed during unfolding/folding by Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) at peptide resolution and by additional biochemical techniques. We found that WT and D93N native hexamers present a stable core and a flexible periphery, the latter being more flexible for the destabilized mutant. Stable but inactive species formed during unfolding of D93N and folding of WT were characterized. For the first time, we show that both of these species are native-like dimers, each of its monomers having a major subdomain folded, while a minor subdomain (Kpn/α 0 ) remains unfolded. The Kpn/α 0 subdomain, which belongs to the catalytic site, becomes structured only upon hexamerization, explaining why oligomerization is required for NDPK activity. Further HDX-MS studies are necessary to establish the general activation mechanism for other homo-oligomers.

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HDX-MS guided drug discovery: small molecules and biopharmaceuticals

Cited by 82 publications

References 82 publications

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

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