Studying conformational dynamics and molecular recognition using integrated structural biology in solution Michael Sattler

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1 Studying conformational dynamics and molecular recognition using integrated structural biology in solution Michael Sattler

2 Outline Dynamics in RNA recognition by multidomain RNA binding proteins Role of weak domain-domain interactions and domain linkers Conformational ensembles from combining NMR with SAXS/SANS Dynamics from combining NMR with FRET, SAXS and EPR

3 Posttranscriptional regulation of gene expression mrna processing: regulatory cis elements (RNA sequence motifs) in mrna trans-acting factors: multi-domain RNA binding proteins (RBPs) RBP start coding sequence (CDS) stop intron 5 m 7 G exon exon exon exon AAAAAA 3 intron cap 5 UTR 3 UTR poly-a tail cis elements Nature (2013) RBPs Cell (2012) Ray et al Hughes, Nature 2013 Baltz, et al Landthaler, Mol Cell 2012; Castello, et al Hentze, Cell 2012

4 RNA recognition by multidomain RNA binding proteins Conformational dynamics: multi-domain RBPs Mackereth et al, Nature (2011) RNA binding protein binding Cooperativity: multiple RBPs RNA binding protein 1 RNA binding protein 2 Hennig, et al, Nature (2014)

5 Combining NMR and SAXS/SANS for studying multi-domain protein complexes in solution Subunit-selective/segmental labeling Sortase A ligation, optimized 2 H-labeling sensitivity, spectral simplification Conformational dynamics: NMR relaxation Domain interfaces/intramolecular contacts Chemical shift perturbations (CSP) PREs (spin labeling): ~ 1/r 6, <20Å Solvent PREs PRE (spin label) Solvent PREs Domain arrangements Residual dipolar couplings (RDCs) Small angle scattering (SAS) RDCs SAXS/SANS Simon, et al Angew. Chem (2010) ; Madl et al JACS (2010) ; Madl et al Angew. Chem (2011); Madl et al J Struct Biol (2011)

6 Small angle X-ray/neutron scattering I( s) A ( s) A ( s) A ( s) a s s particle solvent hydration shell b b 2 molecular weight, radius of gyration, shape in solution Validate structural models characterize conformational dynamics and equilibria Mertens & Svergun J Struct Biol 2010

7 Representation of SAS data SAXS curve Guinier aggregation SAXS curve Kratky flexibility

8 SAXS data Fourier transformation P(r) shape Mertens & Svergun J Struct Biol 2010

9 3 splice site recognition by U2AF spliceosome assembly involves multidomain RBP/RNA interactions Complex E early spliceosome assembly Py tract 3 ss

10 Multidomain arrangements from NMR data Individual domain structures available Spin labeling (e - ) induces paramagnetic relaxation enhancements (PRE) PRE-derived distance restraints to define interdomain arrangement Interdomain distance restraints Spin label Simon, et al (2010) Angew. Chem; Madl, et al (2010) JACS

11 RNA binding by U2AF65 involves a conformational shift Bound to U9 RNA No RNA RRM1 residue RRM2 RRM1 residue RRM2 RRM2 RRM1 open active closed inactive RRM1 RRM2 Mackereth et al Nature (2011)

12 Solution conformation differs from crystal structure NMR Importance of using solution methods for studying multidomain proteins RRM1 RRM2 RDC exp [Hz] RRM1 RRM2 RDC calc [Hz] X-ray RRM1 RNA RRM2 RRM1 RRM2 RRM1+2 linker RDC exp [Hz] Sickmier et al Mol. Cell (2006) RDC calc [Hz]

13 Free U2AF65 samples non-compact conformations Small Angle Scattering data indicate non-compact conformations in free RRM1,2 free RRM1,2 is an ensemble of compact and non-compact states In contrast, RRM1,2/RNA is compact SAXS P(r) Pair distribution function (a.u.) RRM1-RRM2/RNA (compact) RRM1-RRM2 (compact & non-compact states) detached/ non-compact r [Å]

14 Ensemble of RRM1,2 based on NMR and SAS data Ensemble with randomized linker Unbiased/unrestrained selection of conformations and populations Monte-Carlo based error analysis active PRE Center-of-mass of RRM2: full ensemble (pool) selected closed open cross validation passive RRM1 RRM1 RDC SAXS experiment calculated Huang et al JACS (2014)

15 NMR and SAXS define the RRM1,2 solution ensemble Center of mass on RRM1 Pool Distance from COM (RRM1) 0 ~50% of conformations are encounter like electrostatic interactions RRM2 ensemble RRM1 Selected (NMR & SAXS) Distance from COM (RRM1) 0 RRM2 0 Distance from COM (RRM1) Huang et al JACS (2014)

16 Modulation of encounter-like domain interactions PRE for spin-labeled A318C RRM1,2 at different salt concentrations Encounter-like charged interactions are salt dependent 0 mm, 50 mm, 150 mm, 200 mm Huang et al JACS (2014)

17 Unbound U2AF65 is a dynamic ensemble of inactive states Dynamic ensemble of inactive states favorable conformational entropy Consistent with fly-casting mechanism for RNA binding closed inactive RRM2 SAXS open active RRM2 RRM1 Huang et al Sattler, Blackledge, JACS (2014)

18 Crystal structure of the ternary SXL-CSD1-18mer complex Translational regulation in dosage compensation Binding of Sxl and Unr to mrna SXL-RRM1 Non-canonical SXL-RNA contacts msl2 mrna SXL-RRM2 UNR-CSD1 2.8 Å resolution triple zipper contacts Hennig, et al, Nature (2014)

19 Structure validation in solution by NMR UNR-CSD1 48 UNR-CSD Ternary triple zipper contacts

20 Contrast matching with SANS 49 Small Angle Neutron Scattering (SANS) with solvent H 2 O/D 2 O contrast variation protein matching (42% D2O) RNA matching (70% D2O)

21 Contrast matching with SANS 50

22 Structure validation in solution by SAXS and SANS 51 SAXS and SANS data fully corroborate the crystal structure in solution MW I(0) = 34.2 kda, expected: 33.5 kda log(i) 1.5 SAXS data mer fit R g = 2.29 nm 2 = Q (Å -1 ) SAXS DAMMIN log(i) SANS data fit 1 H-SXL 2 D-CSD1 1 H-RNA 42 % D 2 O log(i) SANS data fit 1 H-SXL 2 D-CSD1 1 H-RNA 70 % D 2 O SXL UNR f log(i) Q (Å -1 ) 2 = 1.35 SANS data fit SANS 2 D-SXL 1 H-CSD1 1 H-RNA 42 % D 2 O log(i) = Q (Å -1 ) SANS data fit 2 = D-SXL 1 H-CSD1 1 H-RNA 70 % D 2 O 180 RNA MONSA shape = Q (Å -1 ) Q (Å -1 )

23 Joint refinement of NMR and crystallographic data 52 RDC data in ternary complex agree with domain orientation in crystal structure Experimental RDC [Hz] SXL phages HN-N Q RDC = 0.41 D = R = 0.17 SXL 60 Couplings Back calculated RDC [Hz] Joint NMR/X-ray refinement (REFMAC-NMR) Experimental RDC [Hz] CSD1 phages HN-N Q RDC = 0.39 D = R = 0.28 CSD1 31 Couplings Back calculated RDC [Hz] Overall structure unchanged But: alternative conformations of Y164 Collaboration with Luchinat & coworkers; Carlon et al, JACS (2016)

24 Conclusions Structural dynamics of multi-domain RNA binding proteins is important for their functional activity Cooperative binding of multiple RNA binding domains (RBDs) expands the protein-rna interaction network to regulate diverse biological functions with a limited set of RBDs: protein- RNA recognition code (ensemble) RNA binding protein 1 RNA binding protein 2 Integrated structural biology solution techniques, i.e. NMR, SAXS, SANS, FRET, EPR to study conformation and dynamics of multi-domain proteins and complexes

25 Acknowledgements U2AF SXL/UNR Carolina Sanchez Janosch Hennig Lisa Warner Hyun-Seo Kang Katja Barthelmes Ashish Kawale Carolina Lisa Grzegorz Popowicz Arie Geerlof Miriam Sonntag Iren Wang Janosch Miriam Cameron Mackereth SAXS Tobias Madl Peijian Zou Bernd Simon Katja Hanso Grzegorz Arie Collaborations Ralf U2AF: Sophie Bonnal, Juan Valcarcel (CRG Barcelona); Ensembles: Martin Blackledge, Jie-Rong Huang, Frank Gabel (IBS Grenoble); FRET: Lena Voithenberg, Don Lamb (LMU Munich). SXL/UNR: Cristina Militti, Fatima Gebauer (CRG Barcelona)