Structural Basis of Z-disc Protein/Protein Networks and Functional Implications
Final Report Abstract
In phase II of the FOR1352 research cluster, we have focused on the C-terminal part of the filamentous protein obscurin, which connects specific regions of the sarcomere, namely the Z-disk and M-band with neighboring cellular structures, such as the sarcoplasmic reticulum. We have been able to finish the work on the titin-obscurin complex from phase I and succeeded in determining the high-resolution crystal structures of the titin/obscurin Z-disk complex, titin Ig domain Z8, and obscurin tandem Ig domains O58-O59. In addition, we have validated the structures by thorough biophysical and biochemical characterization. We have performed detailed mutagenesis studies in combination with quantitative affinity measurements using isothermal titration calorimetry to characterize the titin/obscurin interaction interfaces. Small angle X-ray scattering of the titin-obscurin complex in solution complemented the X-ray crystal structure as a cross-validation. Furthermore, in vivo Z-disk localization experiments in neonatal rat cardiomyocytes have been performed to support the in vitro results. Protein kinases can function as dynamic molecular switches and regulate a plethora of biological processes. To investigate the roles of the obscurin kinases in signaling events in or near the Z-disk we have cloned a large variety of obscurin constructs containing either of the two kinases. Heterologous expression in various E. coli and baculovirus-infected insect cells expression hosts followed by extensive screening for suitable purification methods have been performed. The obsurin kinase-containing constructs exhibited extraordinary insolubility and instability during the protein production process, resulting in no usable protein samples for structural investigation to date. Therefore, the studies have been expanded to production and characterization of the extended obscurin signaling domains. We have established robust protein production methods for the SH3-DH-PH triad segment and several smaller constructs. In addition, we succeeded in production of the obscurin IQIg62 motif and characterization of the interaction with its partner protein calmodulin using isothermal titration calorimetry. Crystallization aiming at structural characterization of the obscurin signaling domains including complexes with partner proteins is currently in progress. Taken together, our obtained crystal structures and other results contribute to the elucidation of the overall structure and function of the entire Z-disk signaling end of obscurin. The structural information allows for addressing questions about the functional involvement of the individual domains and the protein-protein complex formation in sarcomeric signaling events. The prospect of characterizing these key signaling domains and their proposed roles in the signal transduction of the sarcomere and control of myofibrillogenesis will not only lead to comprehension of diseases related to obscurin but also to the clarification of cellular mechanisms in the muscle.
Publications
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Telethonin deficiency is associated with maladaptation to biomechanical stress in the mammalian heart. Circ Res. 2011 Sep 16;109(7):758-69
Knöll R, Linke WA, Zou P, Miocic S, Kostin S, Buyandelger B, Ku CH, Neef S, Bug M, Schäfer K, Knöll G, Felkin LE, Wessels J, Toischer K, Hagn F, Kessler H, Didié M, Quentin T, Maier LS, Teucher N, Unsöld B, Schmidt A, Birks EJ, Gunkel S, Lang P, Granzier H, Zimmermann WH, Field LJ, Faulkner G, Dobbelstein M, Barton PJ, Sattler M, Wilmanns M, Chien KR
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Molecular basis of the head-to-tail assembly of giant muscle proteins obscurin-like 1 and titin. EMBO Rep. 2010 Jul;11(7):534-40
Sauer F, Vahokoski J, Song YH, Wilmanns M
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Fast-folding alphahelices as reversible strain absorbers in the muscle protein myomesin. Proc Natl Acad Sci USA. 2011 Aug 23;108(34):14139-44
Berkemeier F, Bertz M, Xiao S, Pinotsis N, Wilmanns M, Gräter F, Rief M
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Superhelical architecture of the myosin filament-linking protein myomesin with unusual elastic properties. PLoS Biol. 2012 Feb;10(2):e1001261
Pinotsis N, Chatziefthimiou SD, Berkemeier F, Beuron F, Mavridis IM, Konarev PV, Svergun DI, Morris E, Rief M, Wilmanns M
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Two immunoglobulin tandem proteins with a linking ß-strand reveal unexpected differences in cooperativity and folding pathways. J Mol Biol. 2012 Feb 10;416(1):137-47
Steward A, Chen Q, Chapman RI, Borgia MB, Rogers JM, Wojtala A, Wilmanns M, Clarke J
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Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling. Mol Biol Cell. 2013 Oct;24(20):3215-26
Eulitz S, Sauer F, Pelissier MC, Boisguerin P, Molt S, Schuld J, Orfanos Z, Kley RA, Volkmer R, Wilmanns M, Kirfel G, van der Ven PF, Fürst DO
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Induction of insulin-like growth factor 1 splice forms by subfragments of myofibrillar proteins. Mol Cell Endocrinol. 2015 Jan 5;399:69-77
Kravchenko IV, Furalyov VA, Chatziefthimiou S, Wilmanns M, Popov VO
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Combination of Whole Genome Sequencing, Linkage, and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy With Features of Left Ventricular Noncompaction. Circ Cardiovasc Genet. 2016 Oct;9(5):426-435
Hastings R, de Villiers CP, Hooper C, Ormondroyd L, Pagnamenta A, Lise S, Salatino S, Knight SJ, Taylor JC, Thomson KL, Arnold L, Chatziefthimiou SD, Konarev PV, Wilmanns M, Ehler E, Ghisleni A, Gautel M, Blair E, Watkins H, Gehmlich K
