Applications of MX, SAXS and biophysical methods in the studies of - - PowerPoint PPT Presentation

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Kristina Djinovi -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

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Kristina Djinović-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, 26th October 2017

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Striated Muscle and its Filaments

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Z-disk intercatome

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Z-disk viewed across the longitudinal sarcomeric axis: paracrystalline

Figure credits: Luther 2009 J Muscle Res Cell Motil; Gautel 2010 COCB Under contraction Relaxed Number of cross- links (α-actinins) depends on titin isoforms

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Z-disk interactome: from qualitative description to (ultra)structure via integrative structural biology

From www.e-heart.org NOT TO SCALE

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Aim

Molecular architecture and assembly

  • f the muscle Z-disc
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Z-disk assembly is hierarchical: Towards the global minimum

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FUNCTION STRUCTURE

1011010101000100100 0100110101001010010 0101010010010001001 1010010101010111111 0101010011010100010 1010010010101011111 1001001101011110100

SAXS X ray NMR EM

Computational Biology Cell Biophysics Biochemistry Biophysics Proteomics Crosslinking

In Integrative St Struct ctural Biol

  • log
  • gy
558 560 562 564 m/z 20 40 60 80 100

Approach: Structure Generation

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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
  • Ca2+ binding in non-muscle forms
  • Antiparallel dimer
  • Multivalent mediator of PPI with many cytoskeletal or regulatory

proteins

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α-actinin crosslinks F-actin

Taylor K A et al. J Cell Biol, 2000

Skeletal muscle Smooth muscle Cardiac muscle Dictyostelium discoidium, a nonmuscle isoform

Hampton et al. 2007

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Major Z-disk protein a-actinin-2

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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

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Human muscle α-actinin-2

Rfree = 0.258 / Rwork = 0.208, @3.5 Å

90o Ribeiro et al., Cell 2014

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PIP2 binding site in α-actinin-2

Neck SR1 SR4 EF34 EF12 ABD

MD combined with flexible docking 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

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Non-activatable PIP2 mutant

Mutants: R163E R163E/R169E R163E/R169E/R192E – PIP2 mutant

Neck EF34 EF12

R163

SR1 SR4 ABD

R169 R192

Ribeiro et al., Cell 2014

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Constitutively active mutant (NEECK)

* * * N E E C K V E

NEECK mutant R268E/I269E/L273E / / / / Ribeiro et al., Cell 2014

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Mutants bind to F-actin and titin Zr-7

  • F-Actin Co-Sedimentation assay
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Non-activatable PIP2 mutant

  • PIP2-C16* binding to α-actinin-2
  • MST

PIP2-C16*

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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)
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Structure of NEECK and structural plasticity of α-actinin in solution (SAXS)

MX model: cartoon SAXS model: solvent accessible area Conformation in the Z-disk

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Structural plasticity and dynamics of α-actinin-2 (EPR)

  • Assessed by EPR/DEER and cw EPR

C862-C270 pair distance is 12 Å

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Conformational plasticity of α-actinin-2

PIP2-C16*

EPR/DEER

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Affinity to titin Zr-7 +/-PIP2 (MST)

n PIP2 facilitates interaction with Zr-7 n Conjunction with structural plasticity

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Constitutively active NEECK in vivo

  • NRCs transfection with

pEGFP-N3 α-actinin WT and NEECK mutant

  • Collaboration with M.

Gautel, KCL

NEECK NEECK NEECK WT WT Ribeiro et al., Cell 2014

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Protein dynamics (FRAP)

α-actininclosed α-actininopen

Collaboration with M. Gautel, KCL Ribeiro et al., Cell 2014

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F-actin:a-actinin interaction

Actin Binding Sites in α-actinin mapped on WT structure

ABS

ABS

Ribeiro et al., Cell 2014

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α-actinin on F-actin

ABD:F-actin

Galkin et al., NSMB 2010 Ribeiro et al., Cell 2014

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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

  • PIP2 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

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Mechanics of α-actinin – titin interaction

Collaboration with M. Rief: Grison et al., PNAS 2017

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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?

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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

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Mechanostability of Zr7-EF3-4 interaction

  • Passive-mode time trace of 5 s at an average force
  • f 3.7 pN
  • Dwell times, unbinding events: @ 6 pN, rates: 0.6 s-1
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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, kon, KD = 4 μM ± 2 μM
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Comparison with other titin Zr

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More genuine geometry

  • In muscle force is applied along titin

filament and not α-actinin

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More genuine geometry – new design

  • Force applied only to Zr7 !
  • Dwelling times, unbinding events:

@12 pN, rates: 0.6s-1

  • Competition assay using Zr7: KD of 8 ± 4 μM
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How is the task of firmly anchoring titin within the Z-disk even under applied mechanical loads achieved?

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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

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Acknowledgments

Collaborations:

  • D. Svergun (EMBL)
  • M. Rief (TUM)
  • M. Gautel (KCL)
  • D. Fuerst (U. Bonn)
  • P. Eliott (UCL)
  • K. Gehmlich (UOX)
  • H. Watkin (UOX)
  • B. Zagrovic (UNIVIE)
  • K. Pirker (BOKU)

Funding: FWF-DACH, FP7-ITN, FFG, UNIVIE, WelcomeTrust, CDG Julius Kostan Georg Mlynek Martin Puchlinger Valeria Stefania Joan Lopez Arolas Antonio Sponga Tobias Thoeni Dominic Puehringer Claudia Schreiner Karolina Zielinska Eneda Höllerl Sara Sajko Marco Grison (TUM) Irina Grishkovskaya Euripedes Ribeiro Nikos Pinotsis Alex Charnagalov Eirini Gkougkoulia Anita Salmazo

  • E. Egelman (Virginia)
  • S. Raunser (MPI Dortmund)
  • B. Warscheid (U. Freiburg)

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