Kristina Djinovi ć -Carugo Applications of MX, SAXS and biophysical methods in the studies of sarcomeric proteins Department of Structural and Computational Biology Max F. Perutz Laboratories University of Vienna Austria Bonn, 26 th October 2017
Striated Muscle and its Filaments
Z-disk intercatome
Z-disk viewed across the longitudinal sarcomeric axis: paracrystalline Relaxed Under contraction Number of cross- links ( α -actinins) depends on titin isoforms Figure credits: Luther 2009 J Muscle Res Cell Motil; Gautel 2010 COCB
Z-disk interactome: from qualitative description to (ultra)structure via integrative structural biology From www.e-heart.org NOT TO SCALE
Aim Molecular architecture and assembly of the muscle Z-disc
Z-disk assembly is hierarchical: Towards the global minimum
Approach: Structure Generation STRUCTURE SAXS X ray NMR EM Integrative St In Struct ctural Biol olog ogy 100 1011010101000100100 80 0100110101001010010 60 0101010010010001001 40 1010010101010111111 20 0101010011010100010 0 1010010010101011111 558 560 562 564 1001001101011110100 Proteomics m/z Biochemistry Cell Computational Crosslinking Biophysics Biophysics Biology FUNCTION
The major Z-disk protein a -actinin-2 • Actin binding domain (ABD): 2 CH domains in tandem • ABD - binding site for F-actin • Binding site for PIP2 • Rod domains: 4 spectrin-like repeats • Dimerization and PPI • Flexible neck connecting ABD and rod • Regulatory Calmodulin-like domain (CaM), 4 EF hands • EF3-4 Binding site for titin Z-repeat 7 • Ca 2+ binding in non-muscle forms • Antiparallel dimer • Multivalent mediator of PPI with many cytoskeletal or regulatory proteins
α -actinin crosslinks F-actin Skeletal muscle Smooth muscle Cardiac muscle Hampton et al. 2007 Dictyostelium discoidium, a nonmuscle isoform Taylor K A et al. J Cell Biol, 2000
Major Z-disk protein a -actinin-2
a -actinin-2 regulation by PIP2 CaM binds to titin Zr-1, 3, 7 AND to neck region Titin: α -actinin interaction controlled by a phospholipid-regulated intramolecular pseudoligand mechanism Figure credits: Julius Kostan Young and Gautel, EMBO J 2000
Human muscle α -actinin-2 90 o R free = 0.258 / R work = 0.208, @3.5 Å Ribeiro et al., Cell 2014
PIP 2 binding site in α -actinin-2 MD combined with flexible docking SR1 EF34 ABD Neck SR4 EF12 In 40% out of 10,000 models polar head lands on Arg platform, aliphatic chain(s) extend towards the neck Ribeiro et al., Cell 2014
Non-activatable PIP 2 mutant EF34 ABD SR1 R192 Neck R169 R163 EF12 Mutants: SR4 R163E R163E/R169E R163E/R169E/R192E – PIP2 mutant Ribeiro et al., Cell 2014
Constitutively active mutant (NEECK) / / / / NEECK mutant R268E/I269E/L273E * * * N E E C K V E Ribeiro et al., Cell 2014
Mutants bind to F-actin and titin Zr-7 • F-Actin Co-Sedimentation assay
Non-activatable PIP 2 mutant • PIP2-C16* binding to α -actinin-2 • MST PIP2-C16*
What is the structure of NEECK? • Neck is unstructured when not bound to CaM (NMR), as also titin Zr-7 • Bigger hydrodynamic radius vs WT (SEC-MALLS)
Structure of NEECK and structural plasticity of α -actinin in solution (SAXS) Conformation in the Z-disk MX model: cartoon SAXS model: solvent accessible area
Structural plasticity and dynamics of α -actinin-2 (EPR) • Assessed by EPR/DEER and cw EPR C862-C270 pair distance is 12 Å
Conformational plasticity of α -actinin-2 EPR/DEER PIP2-C16*
Affinity to titin Zr-7 +/-PIP2 (MST) n PIP2 facilitates interaction with Zr-7 n Conjunction with structural plasticity
Constitutively active NEECK in vivo WT • NRCs transfection with NEECK pEGFP-N3 α-actinin WT and NEECK mutant NEECK • Collaboration with M. Gautel, KCL WT NEECK Ribeiro et al., Cell 2014
Protein dynamics (FRAP) α-actinin closed α-actinin open Collaboration with M. Gautel , KCL Ribeiro et al., Cell 2014
F-actin: a -actinin interaction Actin Binding Sites in α -actinin mapped on ABS WT structure ABS Ribeiro et al., Cell 2014
α -actinin on F-actin ABD:F-actin Galkin et al., NSMB 2010 Ribeiro et al., Cell 2014
α -actinin binding to F-actin and titin in Z-disk Distance between filaments in agreement with distance in tetragonal Z-disk lattice (200 Å) Implications also for non-muscle isoforms Dynamics and structural plasticity are essential for function and regulation • PIP 2 increases affinity for interaction with Zr-7 • Structural flexibility required for regulation and function: binding F-actin and titin Movie credits: N. Pinotsis Ribeiro et al., Cell 2014
Mechanics of α -actinin – titin interaction Collaboration with M. Rief: Grison et al., PNAS 2017
Mechanics of α -actinin – titin interaction • Affinities of titin Z-repeats to EF3-4 of α -actinin in micromolar range • Forces under muscle contraction: 200 pN • Question : • How is the task of firmly anchoring titin within the Z-disk even under applied mechanical loads achieved?
Mechanostability of Zr7-EF3-4 interaction • Optical tweezers: single molecule mechanics • Probe the mechanical strength of interaction • Force propagates both through the α -actinin EF3-4 and Zr7 Grison et al, 2017, PNAS
Mechanostability of Zr7-EF3-4 interaction • Passive-mode time trace of 5 s at an average force of 3.7 pN • Dwell times, unbinding events: @ 6 pN, rates: 0.6 s -1
Mechanostability of Zr7-EF3-4 interaction • To determine binding rates and the dissociation constant : • Competition assay with free titin Zr-7 peptide • New state: peptide from solution bound, blocking rebinding of tethered Zr7 • k off, k on, K D = 4 μ M ± 2 μ M
Comparison with other titin Zr
More genuine geometry • In muscle force is applied along titin filament and not α -actinin
More genuine geometry – new design • Force applied only to Zr7 ! • Dwelling times, unbinding events: @12 pN, rates: 0.6 s -1 • Competition assay using Zr7: K D of 8 ± 4 μ M
How is the task of firmly anchoring titin within the Z-disk even under applied mechanical loads achieved?
Model: Avidity at Work • Forces on a single titin molecule: cca 5 pN • Midpoint force: 3.5 pN • Midpoint force: 13 pN • Kinetic data: residing times on titin a few sec • 4 titin Zr bind to α -actinin: avidity effect • Interaction free energies sum-up due to the increased valency • Dynamic bonds acting together lead to a long-term stable anchor Grison et al, 2017, PNAS
Acknowledgments Present: Past: Julius Kostan Dominic Puehringer Euripedes Ribeiro Tamas Hatfaludi Georg Mlynek Claudia Schreiner Nikos Pinotsis Adekunle Onipe Martin Puchlinger Karolina Zielinska Alex Charnagalov Eduardo Bezerra Valeria Stefania Eneda Höllerl Eirini Gkougkoulia Ariadna R. Chamorro Joan Lopez Arolas Sara Sajko Anita Salmazo Jae-Geun Song Antonio Sponga Marco Grison (TUM) Vid Puz Tobias Thoeni Irina Grishkovskaya Bjoern Sjoeblom Funding : Collaborations : E. Egelman (Virginia) FWF-DACH, FP7-ITN, D. Svergun (EMBL) FFG, UNIVIE, WelcomeTrust, CDG M. Rief (TUM) S. Raunser (MPI Dortmund) B. Warscheid (U. Freiburg) M. Gautel (KCL) D. Fuerst (U. Bonn) Evitra (Stockholm) P. Eliott (UCL) K. Gehmlich (UOX) H. Watkin (UOX) B. Zagrovic (UNIVIE) K. Pirker (BOKU)
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