Structure and Regulation of the Myofibrillar Z-disc Interactome

• Antragstellerin: Professorin Dr. Kristina Djinovic-Carugo
• Fachliche Zuordnung: Strukturbiologie
• Förderung: Förderung von 2010 bis 2018
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 148688621

• Erstellungsjahr: 2019

Zusammenfassung der Projektergebnisse

In the course of the second funding period the research activities were focused on the understanding of the impact of posttranslational modifications in filamin C domains 19-21, on the interaction with selected binding partners (FATZ-1 and Pgm5/aciculin), structural analysis of complexes of FATZ-1 with filamin C and α-actinin-2, and on the structural and biochemical analysis of PGM5/aciculin. Comparative biophysics analysis of FlnC d19-21 and its phosphomimetic variants (S2233D S2236D) showed notably increased capacity of the modified protein to refold after thermal unfolding, compared to the wild-type construct. Being thermal unfolding a proxy to the mechanical stress and unfolding, we hypothesize this might be attributed to the increased capacity of the protein to recover from mechanical strain and mechanical damage during muscle contraction. Analysis of binding affinities of FlnC d19-21 and its phosphomimetic mutants to FATZ-1 variants showed that phosphorylation indeed regulated the affinity of FlnC to FATZ-1, namely it abrogates of of the two interaction sites with the N-ter region of FATZ-1. On the other hand, a single phosphomimetic mutation in FlnC d19-21 does not impact on the interaction with PGM5/aciculin. We further found that FlnC and α-actinin-2 compete for the biding site on FATZ-1, located in the C-terminal portion of the protein, where α-actinin-2 displaces FlnC, leading to formation of a ternary complex between FlnC, α-actinin-2 and FATZ-1. Crystal structure determination of FlnC d21 and α-actinin-2 rod in complex with FATZ-1 for the first time allowed to visualize the mode of interaction at the atomic level. In effect, these are the first crystal structures of intrinsically disordered Z-disk protein FAZT-1 with two of its major binding partners FlnC and α-actinin-2. PGM5/aciculin was initially not part of the research plans, but since PI1 discovered PGM5/aciculin binds several Z-disk proteins, we embarked on its structural and biochemical characterization. In the course of the binding studies we found that PGM5/aciculin binds FATZ-1, which was not shown before. Additionally, we showed that PGM5/aciculin does possess an enzymatic activity of phosphoglucomutase, which changes the paradigm regarding PGM5/aciculin's role in the Z-disk, from a purely structural protein to a scaffolding enzyme potentially regulated by interaction partners. We thus determine its crystal structure which unexpectedly revealed notable differences in the conformation of active site. These results are a solid basis for a number of follow-up experiments both on structural as well as biochemical level to understand the mechanism of action and its potential regulation by Z-disk binding partners.

Projektbezogene Publikationen (Auswahl)

• Opening of tandem calponin homology domains regulates their affinity for F-actin. Nat Struct Mol Biol 2010; 17:614-616
  Galkin VE, Orlova A, Salmazo A, Djinović-Carugo K, Egelman EH
  
• Structural Portrait of Filamin Interaction Mechanisms. Curr Protein Pept Sci 2010; 11: 639-650
  Djinović-Carugo K, Carugo O
  
• Mutations in the N- terminal actin-binding domain of filamin C cause a distal myopathy. Am J Hum Genet 2011; 88: 729-740
  Duff RM, Tay V, Hackman P, Ravenscroft G, McLean C, Kennedy P, Steinbach A, Schöffler W, van der Ven PFM, Fürst DO, Song J, Djinović-Carugo K, Penttila S, Raheem O, Reardon K, Malandrini A, Gambelli S, Villanova M, Nowak KJ, Williams DR, Landers JE, Brown RHJr, Udd B, Laing NG
  
• Pathophysiology of protein aggregation and extended phenotyping in filaminopathy. Brain 2012; 135: 2642-2660
  Kley RA, Serdaroglu-Oflazer P, Leber Y, Odgerel Z, van der Ven PFM, Olive M, Ferrer I, Onipe A, Mihaylov M, Bilbao JM, Lee HS, Höhfeld J, Djinović-Carugo K, Kong K, Tegenthoff M, Peters SA, Stenzel W, Vorgerd M, Goldfarb LG, Fürst DO
  
• Myopodin is an F-actin bundling protein with multiple independent actin-binding regions. J Musc Res Cell Motil 2013; 34: 61-69
  Linnemann A, Vakeel P, Bezerra E, Orfanos Z, Djinović-Carugo K, van der Ven PFM, Kirfel G, Fürst DO
  
• Direct interaction of actin filaments with F-BAR protein pacsin2. EMBO Rep 2014; 15: 1154-1162
  Kostan J, Salzer U, Orlova A, Toro I, Hodnik V, Senju Y, Zou J, Schreiner C, Steiner J, Merilainen J, Nikki M, Virtanen I, Carugo O, Rappsilber J, Lappalainen P, Lehto VP, Anderluh G, Egelman EH, Djinović-Carugo K
  
• The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes. Amino Acids 2014; 46: 1565-1582
  Mlynek G, Lehner A, Neuhold J, Leeb S, Kostan J, Charnagalov A, Stolt-Bergner P, Djinović-Carugo K, Pinotsis N
  
• The structure and regulation of human muscle alpha-actinin. Cell 2014; 159: 1447-1460
  Ribeiro EA Jr, Pinotsis N, Ghisleni A, Salmazo A, Konarev PV, Kostan J, Sjoblom B, Schreiner C, Polyansky AA, Gkougkoulia EA, Holt MR, Aachmann FL, Zagrovic B, Bordignon E, Pirker KF, Svergun DI, Gautel M, Djinović-Carugo K
  
• Structural Insights into Ca- Calmodulin Regulation of Plectin 1a-Integrin beta4 Interaction in Hemidesmosomes. Structure 2015; 23: 1-13
  Song JG, Kostan J, Drepper F, Knapp B, de Almeida Ribeiro EJr, Konarev PV, Grishkovskaya I, Wiche G, Gregor M, Svergun DI, Warscheid B, Djinović-Carugo K
  
• Congenital macrothrombocytopenia-linked mutations in the actin-binding domain of alpha-actinin-1 enhance F-actin association. FEBS Lett 2016; 590: 685-695
  Murphy AC, Lindsay AJ, McCaffrey MW, Djinović-Carugo K, Young PW
  
• Structure and calcium-binding studies of calmodulinlike domain of human non-muscle alpha-actinin-1. Sci Rep 2016; 6: 27383
  Drmota PS, Slapsak U, Pavsic M, Ilc G, Puz V, de Almeida Ribeiro E, Anrather D, Hartl M, Backman L, Plavec J, Lenarcic B, Djinović-Carugo K
  
• The sarcomeric cytoskeleton: from molecules to motion. J Exp Biol 2016; 219: 135-145
  Gautel M, Djinović-Carugo K
  
• alpha-actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk. Proc Natl Acad Sci USA 2017; 114: 1015-1020
  Grison M, Merkel U, Kostan J, Djinović-Carugo K, Rief M
  
• Conformational plasticity and evolutionary analysis of the myotilin tandem Ig domains. Sci Rep 2017; 7: 3993
  Puž V, Pavsic M, Lenarcic B, Djinović-Carugo K
  
• Deciphering the BAR code of membrane modulators. Cell Mol Life Sci 2017; 74: 2413-2438
  Salzer U, Kostan J, Djinović-Carugo K
  
• Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered. J Gen Vir 2017; 98: 471-485
  Rieder FJJ, Kastner MT, Hartl M, Puchinger MG, Schneider M, Majdic O, Britt WJ, Djinovic-Carugo K, Steininger C
  
• HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C. Sci Adv 2019; 5: eaav8421
  Collier MP, Alderson TR, de Villiers CP, Nicholls D, Gastall HY, Allison TM, Degiacomi MT, Jiang H, Mlynek G, Fürst DO, van der Ven PFM, Djinović-Carugo K, Baldwin AJ, Watkins H, Gehmlich K, Benesch JLP