Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants

The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid–protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7×108 M−1). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid–water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholinebinding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation

Protein Thermodynamic Destabilization in the Assessment of Pathogenicity of a Variant of Uncertain Significance in Cardiac Myosin Binding Protein C.

In the era of next generation sequencing (NGS), genetic testing for inherited disorders identifies an ever-increasing number of variants whose pathogenicity remains unclear. These variants of uncertain significance (VUS) limit the reach of genetic testing in clinical practice. The VUS for hypertrophic cardiomyopathy (HCM), the most common familial heart disease, constitute over 60% of entries for missense variants shown in ClinVar database. We have studied a novel VUS (c.1809T>G-p.I603M) in the most frequently mutated gene in HCM, MYBPC3, which codes for cardiac myosin-binding protein C (cMyBPC). Our determinations of pathogenicity integrate bioinformatics evaluation and functional studies of RNA splicing and protein thermodynamic stability. In silico prediction and mRNA analysis indicated no alteration of RNA splicing induced by the variant. At the protein level, the p.I603M mutation maps to the C4 domain of cMyBPC. Although the mutation does not perturb much the overall structure of the C4 domain, the stability of C4 I603M is severely compromised as detected by circular dichroism and differential scanning calorimetry experiments. Taking into account the highly destabilizing effect of the mutation in the structure of C4, we propose reclassification of variant p.I603M as likely pathogenic. Looking into the future, the workflow described here can be used to refine the assignment of pathogenicity of variants of uncertain significance in MYBPC3.J.A.C. was funded by the Ministerio de Ciencia, Innovación y Universidades (MCNU) through grants BIO2017-83640-P (AEI/FEDER, UE) and RYC-2014-16604, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, MINOTAUR, AC16/00045), the Comunidad de Madrid (P2018/NMT-4443) and the CNIC-Severo Ochoa intramural grant program (03-2016 IGP). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). G.F. was funded by the Ministero dell’Istruzione, dell’Università e della Ricerca-Rome PS35-126/IND.S

Structure prediction of honey bee vitellogenin: a multi-domain protein important for insect immunity

Vitellogenin (Vg) has been implicated as a central protein in the immunity of egg-laying animals. Studies on a diverse set of species suggest that Vg supports health and longevity through binding to pathogens. Specific studies of honey bees (Apis mellifera) further indicate that the vitellogenin (vg) gene undergoes selection driven by local pathogen pressures. Determining the complete 3D structure of full-length Vg (flVg) protein will provide insights regarding the structure–function relationships underlying allelic variation. Honey bee Vg has been described in terms of function, and two subdomains have been structurally described, while information about the other domains is lacking. Here, we present a structure prediction, restrained by experimental data, of flVg from honey bees. To achieve this, we performed homology modeling and used AlphaFold before using a negative-stain electron microscopy map to restrict, orient, and validate our 3D model. Our approach identified a highly conserved Ca2+-ion-binding site in a von Willebrand factor domain that might be central to Vg function. Thereafter, we used rigid-body fitting to predict the relative position of high-resolution domains in a flVg model. This mapping represents the first experimentally validated full-length protein model of a Vg protein and is thus relevant for understanding Vg in numerous species. Our results are also specifically relevant to honey bee health, which is a topic of global concern due to rapidly declining pollinator numbers.publishedVersio

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction

Mechanical unfolding of long human telomeric RNA (TERRA)

[EN] We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplex blocks, which unfold at lower forces than their DNA counterparts.This work was supported by grants from the Spanish Ministry of Science and Innovation (grants RYC2007-01765 to JRA-G, BFU2011-30295-C02-01 to AV, and CTQ2010-21567-C02-02 to CG). MG was supported by the FPI fellowship BES-2009-027909. RB and EH-G were supported by Comunidad de Madrid, grant CAM-S2009MAT-1507. AV acknowledges an institutional grant from the Fundacion Ramon Areces to the CBMSO. JRA-G wants to thank Prof. J. L. Carrascosa and Prof. J. M. Valpuesta (CNB-CSIC) for their continuous support and encouragement in this research. We also acknowledge the excellent technical assistance of Beatriz de Pablos (CBMSO).Garavís, M.; Bocanegra, R.; Herrero-Galán, E.; González, C.; Villasante, A.; Arias-Gonzalez, JR. (2013). Mechanical unfolding of long human telomeric RNA (TERRA). Chemical Communications. 49(57):6397-6399. https://doi.org/10.1039/c3cc42981dS639763994957De Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes & Development, 19(18), 2100-2110. doi:10.1101/gad.1346005Blackburn, E. H. (1991). Structure and function of telomeres. Nature, 350(6319), 569-573. doi:10.1038/350569a0Biffi, G., Tannahill, D., McCafferty, J., & Balasubramanian, S. (2013). Quantitative visualization of DNA G-quadruplex structures in human cells. Nature Chemistry, 5(3), 182-186. doi:10.1038/nchem.1548Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D., & Lipps, H. J. (2005). Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. 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Molecular and Cellular Biology, 30(20), 4808-4817. doi:10.1128/mcb.00460-10Deng, Z., Norseen, J., Wiedmer, A., Riethman, H., & Lieberman, P. M. (2009). TERRA RNA Binding to TRF2 Facilitates Heterochromatin Formation and ORC Recruitment at Telomeres. Molecular Cell, 35(4), 403-413. doi:10.1016/j.molcel.2009.06.025De Silanes, I. L., d’ Alcontres, M. S., & Blasco, M. A. (2010). TERRA transcripts are bound by a complex array of RNA-binding proteins. Nature Communications, 1(1). doi:10.1038/ncomms1032Xu, Y., Suzuki, Y., Ito, K., & Komiyama, M. (2010). Telomeric repeat-containing RNA structure in living cells. Proceedings of the National Academy of Sciences, 107(33), 14579-14584. doi:10.1073/pnas.1001177107Martadinata, H., & Phan, A. T. (2009). Structure of Propeller-Type Parallel-Stranded RNA G-Quadruplexes, Formed by Human Telomeric RNA Sequences in K+Solution. Journal of the American Chemical Society, 131(7), 2570-2578. doi:10.1021/ja806592zXu, Y., Kaminaga, K., & Komiyama, M. (2008). G-Quadruplex Formation by Human Telomeric Repeats-Containing RNA in Na+Solution. Journal of the American Chemical Society, 130(33), 11179-11184. doi:10.1021/ja8031532Collie, G. W., Haider, S. M., Neidle, S., & Parkinson, G. N. (2010). A crystallographic and modelling study of a human telomeric RNA (TERRA) quadruplex. Nucleic Acids Research, 38(16), 5569-5580. doi:10.1093/nar/gkq259Collie, G. W., Parkinson, G. N., Neidle, S., Rosu, F., De Pauw, E., & Gabelica, V. (2010). Electrospray Mass Spectrometry of Telomeric RNA (TERRA) Reveals the Formation of Stable Multimeric G-Quadruplex Structures. Journal of the American Chemical Society, 132(27), 9328-9334. doi:10.1021/ja100345zMartadinata, H., Heddi, B., Lim, K. W., & Phan, A. T. (2011). Structure of Long Human Telomeric RNA (TERRA): G-Quadruplexes Formed by Four and Eight UUAGGG Repeats Are Stable Building Blocks. Biochemistry, 50(29), 6455-6461. doi:10.1021/bi200569fArora, A., & Maiti, S. (2009). 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Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.J.A.C. acknowledges funding from the Ministerio de Ciencia e Innovación (MCIN) through grants BIO2014– 54768-P, BIO2017–83640-P (AEI/FEDER, UE), EIN2019–102966, RYC-2014–16604, and BFU2017–90692­ REDT, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, MINOTAUR, AC16/00045), and the Comunidad de Madrid (consortium Tec4Bio-CM, S2018/NMT-4443, FEDER). This work was supported by NIH grants RM1 GM33289 and HL117138 to J.A.S.; a Stanford Dean’s Postdoctoral Fellowship to D.P. and N.N.; and a Stanford Maternal and Child Health Research Institute (MCHRI) Postdoctoral Fellowship (1220552–140-DHPEU) to N.N. Financial support to D.D.S. comes from Eusko Jaurlaritza (Basque Government) through the project IT1254–19, and grants RYC-2016–19590 and PGC2018–099321-B-I00 from the MCIN (FEDER). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCIN, and the Pro CNIC Foundation and was a Severo Ochoa Center of Excellence (SEV-2015–0505). We acknowledge funding from ISCIII to the Centro de Investigación Biomédica en Red (CIBERCV), CB16/11/00425. C.S.C. is the recipient of an FPI-SO predoctoral fellowship, BES-2016–076638. M.R.P. was the recipient of a Ph.D. fellowship from the Italian Ministry of Education, Universities and Research (MIUR). C.P.L. was a recipient of a CNIC Master Fellowship. We thank N. Vicente for excellent technical support (through grant PEJ16/MED/TL-1593 from Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid and the European Social Fund). We thank the Spectroscopy and Nuclear Magnetic Resonance Core Unit at CNIO for access to CD instrumentation and discussion about protein binding assays. We thank A. Thompson and S. Day for their insights. We thank all members of the Molecular Mechanics of the Cardiovascular System team for helpful discussions and the contribution of five anonymous reviewers.S

Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease.This work was supported by the Ministerio de Ciencia e Innovación grants BIO2014-54768-P, BIO2017-83640-P, RYC-2014-16604 to JAC and PGC2018-097019-B-I00 to JV, the Regional Government of Madrid grants S2018/NMT-4443 and PEJ16/MED/TL-1593 to JAC and the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria grant PRB3 (PT17/0019/0003- ISCIII-SGEFI /ERDF, ProteoRed), and “la Caixa” Banking Foundation (project code HR17-00247) to JV. We acknowledge funding from the European Research Area Network on Cardiovascular Disease through grant MINOTAUR to SS (The Austrian Science Fund – FWF, I3301) and JAC (ISCIII-AC16/00045). The CNIC is supported by ISCIII, the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation, and was a Severo Ochoa Center of Excellence (SEV-2015-0505). IMM was the recipient of a CNIC-ACCIONA Masters Fellowship and holds a fellowship from “La Caixa” Foundation (ID 100010434, fellowship code LCF/BQ/DR20/11790009). CSC is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. We thank Wolfgang A. Linke and Pablo García-Pavía for critical feedback. We are also thankful for the insights of three anonymous reviewers.S

Estudio de la hirsutelina como nuevo miembro de la familia de las ribotoxinas

Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular I, leída el 03-07-2008Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

Redox regulation of protein nanomechanics in health and disease: Lessons from titin

The nanomechanics of sarcomeric proteins is a key contributor to the mechanical output of muscle. Among them, titin emerges as a main target for the regulation of the stiffness of striated muscle. In the last years, single-molecule experiments by Atomic Force Microscopy (AFM) have demonstrated that redox posttranslational modifications are strong modulators of the mechanical function of titin. Here, we provide an overview of the recent development of the redox mechanobiology of titin, and suggest avenues of research to better understand how the stiffness of molecules, cells and tissues are modulated by redox signaling in health and disease

Solution structure of hirsutellin A - New insights into the active site and interacting interfaces of ribotoxins

10 pags, 4 figsHirsutellin (HtA) is intermediate in size between other ribotoxins and less specific microbial RNases, and thus offers a unique chance to determine the minimal structural requirements for activities unique to ribotoxins. Here, we have determined the structure of HtA by NMR methods. The structure consists of one α-helix, a helical turn and seven β-strands that form an N-terminal hairpin and an anti-parallel β-sheet, with a characteristic α + β fold and a highly positive charged surface. Compared to its larger homolog α-sarcin, the N-terminal hairpin is shorter and less positively charged. The secondary structure elements are connected by large loops with root mean square deviation (rmsd) values > 1 Å, suggesting some degree of intrinsically dynamic behavior. The active site architecture of HtA is unique among ribotoxins. Compared to α-sarcin, HtA has an aspartate group, D40, replacing a tyrosine, and the aromatic ring of F126, located in the leucine 'environment' close to the catalytic H113 in a similar arrangement to that found in RNase T1. This unique active site structure is discussed in terms of its novel electrostatic interactions to understand the efficient cytotoxic activity of HtA. The contributions of the N-terminal hairpin, loop 2 and loop 5 with regard to protein functionality, protein-protein and protein-lipid interactions, are also discussed. The truncation and reduced charge of the N-terminal hairpin in HtA may be compensated for by the extension and new orientation of its loop 5. This novel orientation of loop 5 re-establishes a positive charge on the side of the molecule that has been shown to be important for intermolecular interactions in ribotoxins. © 2009 FEBS.This paper was supported by projects GRICES-CSIC 2007-2008, BFU2005-01855/BMC and BFU2006-04404 of the Spanish Ministerio de Educación y Ciencia, and SFRH/B/35992/2007 of the Portuguese Science and Technology Foundatio