Analysis of structural dynamics by H/D-exchange coupled to mass spectrometry HDX-MS

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1 Analysis of structural dynamics by H/D-exchange coupled to mass spectrometry () New Approaches in Drug Design & Discovery th of March 2014

2 Introduction What are the challenges in structure-based drug design? Crystallographic snap-shots - Water structure - Flexibility - Conformational changes - Allosteric effects target drug drug lock-and-key Fischer, 1894 induced fit Koshland,1958 Alternative NMR (size, aggregation, micelles, labeling) Hydrogen/Deuterium exchange (HDX) conformational dynamics

3 HDX fundamentals What is HDX? Exposure to D 2 O leads to replacement of exchangeable hydrogen atoms. ~1 Da mass difference - read out by mass spectrometry Secondary structure is stabilized by backbone N H O=C hydrogen bonds Other factors: ph, temperature, solvent accessibility Engen, 2009

4 HDX work-flow What do we need to consider experimentally? ph and temperature control! time aspect important! e.g. continuous labeling Brier and Engen, 2010

5 HDX data analysis What are the challenges? complexity, assignment, interpretation Hexicon 2 Lindner et al., JASMS, 2014

6 Biological question blue light photoreceptors What systems are we focusing on? BLUF sensors of Blue Light Using FAD Modular domains Occur as isolated domains but also in complex How is the signal integrated? h dark adapted state signaling inactive light-induced state signaling active BLUF EAL (BlrP1) (In)activation of an enzymatic or another biological function

7 BlrP1* (Klebsiella pneumoniae) Blue light *Blue light regulated Phosphodiesterase 1 BLUF FAD EAL Mg 2+ (not Ca 2+ ) Light signal degradation of second messenger c-di-gmp 5 pgpg Expression of adhesion factors Biofilm formation Sessility Virulence gene expression Single cell dispersal Motility

8 BlrP1 structure Structure solved - BLUF in dark state - c-di-gmp bound - Bimetallic enzyme - Dimer formation How does light influence EAL activity? Barends et al., Nature, 2009

9 BlrP1 structure -barrel -helix Schematic build-up of the EAL dimer Shorten one helix αβ-barrel (TIM-barrel) ½ helix Space for ½ helix Build up a compound helix: compound helix Dimer helices at interface

10 BLUF EAL interface

11 EAL activity c-di-gmp Mn 2+ /ph 9 complex, 2.6 Å resolution In-line attack of activated water (W1) onto P Metal to metal distances correlate with activity! 2.3 A 3.5 A 3.2 A 3.2 A 2.2 A 2.3 A 3.6 A 2.4 A 4.4 A M 4.3 A M M 3.7 A M M 5.3 A M ph 6, low activity, 2.1 Å ph 9, high activity, 2.6 Å ph 8, Ca 2+ inhibited, 2.3 Å

12 EAL activity How can this be regulated by the BLUF domain? Subtle changes sufficient for ~4-fold increase in activity Metal site 2 optimization by light activation? Look at the BLUF EAL interface; which parts of BLUF are in contact? - Substrate bound EAL active? - Dark adapted BLUF inactive? and solution scattering studies

13 BlrP1 A B C D Experiments: BlrP1 Mg 2+, dark BlrP1 Mg 2+, blue-light BlrP1 Ca 2+, c-di-gmp, dark BlrP1 Ca 2+, c-di-gmp, light A B C D combinatorial system c-di-gmp flavin Illumination or substrate binding c-di-gmp flavin C - A Substrate binding

14 BlrP1 Winkler et al., JMB, 2013

15 BlrP1 C - A A B C D Experiments: BlrP1 Mg 2+, dark BlrP1 Mg 2+, blue-light BlrP1 Ca 2+, c-di-gmp, dark BlrP1 Ca 2+, c-di-gmp, light #D ~300 ion series analyzed (multiple charge states) Deuteration time ~100 peptides compared (common to all experiments) Triplicates for 5 timepoints #D Sequence coverage: 95 % Deuteration time

16 BlrP1 HDX results A B C D Experiments: BlrP1 Mg 2+, dark BlrP1 Mg 2+, blue-light BlrP1 Ca 2+, c-di-gmp, dark BlrP1 Ca 2+, c-di-gmp, light B - A D - C C - A D - B sensor substr. bind. dimer interface

17 HDX - results A B C D Experiments: BlrP1 Mg 2+, dark BlrP1 Mg 2+, blue-light BlrP1 Ca 2+, c-di-gmp, dark BlrP1 Ca 2+, c-di-gmp, light C - A B - A D - B substrate binding illumination of free BlrP1 substrate binding in light-state 15 s 1 h 5 min Allostery

18 HDX - results A B C D Experiments: BlrP1 Mg 2+, dark BlrP1 Mg 2+, blue-light BlrP1 Ca 2+, c-di-gmp, dark BlrP1 Ca 2+, c-di-gmp, light C - A substrate binding (dark) Allostery

19 SAXS data Small angle X-ray scattering experiments. characteristic difference between crystal structure and solution data Winkler et al., JMB, 2013 Effect is reproduced by a BlrP1 normal mode clam-shell opening of EAL dimer reflected in various EAL dimers Different EAL dimer structures

20 SAXS data Solution scattering experiments of light-activation. characteristic difference seen in the intermediate angle regime - suggests inter-domain effects This effect is reproduced by another BlrP1 normal mode correlates with HDX in that no global structural rearrangements occur

21 BLUF summary Combination of HDX with SAXS and crystal structure Suggest opening/closing movement of EAL dimer key element - compound helix hinge at dimerization interface Allosteric bi-directional communication Light-induced structural changes affect this region via the BLUF C-terminal helices Integration of HDX provides important insight also for other photoreceptor systems - PPI Winkler et al., NSMB, 2013 Winkler et al., JMB, 2013

22 summary and perspective Benefits of HDX In solution method; no size limits; population sub-states can be addressed Conformational dynamics for identification of important regions/construct design Identification of elements involved in protein:ligand, protein:protein interaction Future developments Single amino acid resolution ETD fragmentation Extension to membrane protein targets

23 Acknowledgements MPImF Heidelberg Robert Lindner Anikó Udvarhelyi Thomas Barends Udo Heintz Elisabeth Hartmann Jochen Reinstein Robert Shoeman Ilme Schlichting Thank you for your attention! PSI Villigen Andreas Menzel

24 HDX applications What can we learn from HDX experiments? Engen, 2009

25 HDX fundamentals Mechanism of backbone HDX Assumption of closed and open conformations EX1 regime: (correlated) EX2 regime: (uncorrelated) 15 s 1 min 5 min 20 min

26 HDX fundamentals Kinetics of HDX Engen, 2009

27 HDX pepsin cleavage How to assign peptides? MS/MS accurate mass

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