Backbone-independent NMR Resonance Assignments of Methyl Probes in Large Proteins

Overview

Journal Nat Commun

Specialty Biology

Date 2021 Jan 30

PMID 33514730

Citations 18

Authors

Santrupti Nerli

Viviane S De Paula

Andrew C McShan

Nikolaos G Sgourakis

Affiliations

Soon will be listed here.

Abstract

Methyl-specific isotope labeling is a powerful tool to study the structure, dynamics and interactions of large proteins and protein complexes by solution-state NMR. However, widespread applications of this methodology have been limited by challenges in obtaining confident resonance assignments. Here, we present Methyl Assignments Using Satisfiability (MAUS), leveraging Nuclear Overhauser Effect cross-peak data, peak residue type classification and a known 3D structure or structural model to provide robust resonance assignments consistent with all the experimental inputs. Using data recorded for targets with known assignments in the 10-45 kDa size range, MAUS outperforms existing methods by up to 25,000 times in speed while maintaining 100% accuracy. We derive de novo assignments for multiple Cas9 nuclease domains, demonstrating that the methyl resonances of multi-domain proteins can be assigned accurately in a matter of days, while reducing biases introduced by manual pre-processing of the raw NOE data. MAUS is available through an online web-server.

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References

Orts J, Vogeli B, Riek R . Relaxation Matrix Analysis of Spin Diffusion for the NMR Structure Calculation with eNOEs. J Chem Theory Comput. 2015; 8(10):3483-92. DOI: 10.1021/ct3002249. View

East K, Newton J, Morzan U, Narkhede Y, Acharya A, Skeens E . Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics. J Am Chem Soc. 2019; 142(3):1348-1358. PMC: 7497131. DOI: 10.1021/jacs.9b10521. View

Lee W, Tonelli M, Markley J . NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics. 2014; 31(8):1325-7. PMC: 4393527. DOI: 10.1093/bioinformatics/btu830. View

Tugarinov V, Kanelis V, Kay L . Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc. 2007; 1(2):749-54. DOI: 10.1038/nprot.2006.101. View

Yang J, Anishchenko I, Park H, Peng Z, Ovchinnikov S, Baker D . Improved protein structure prediction using predicted interresidue orientations. Proc Natl Acad Sci U S A. 2020; 117(3):1496-1503. PMC: 6983395. DOI: 10.1073/pnas.1914677117. View

Xu Y, Matthews S . MAP-XSII: an improved program for the automatic assignment of methyl resonances in large proteins. J Biomol NMR. 2013; 55(2):179-87. DOI: 10.1007/s10858-012-9700-z. View

Marks D, Hopf T, Sander C . Protein structure prediction from sequence variation. Nat Biotechnol. 2012; 30(11):1072-80. PMC: 4319528. DOI: 10.1038/nbt.2419. View

Doudna J, Charpentier E . Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014; 346(6213):1258096. DOI: 10.1126/science.1258096. View

Chao F, Kim J, Xia Y, Milligan M, Rowe N, Veglia G . FLAMEnGO 2.0: an enhanced fuzzy logic algorithm for structure-based assignment of methyl group resonances. J Magn Reson. 2014; 245:17-23. PMC: 4161213. DOI: 10.1016/j.jmr.2014.04.012. View

10.

Behera S, Dubey A, Chen W, De Paula V, Zhang M, Sgourakis N . Nearest-neighbor NMR spectroscopy: categorizing spectral peaks by their adjacent nuclei. Nat Commun. 2020; 11(1):5547. PMC: 7642304. DOI: 10.1038/s41467-020-19325-4. View

11.

Song Y, DiMaio F, Wang R, Kim D, Miles C, Brunette T . High-resolution comparative modeling with RosettaCM. Structure. 2013; 21(10):1735-42. PMC: 3811137. DOI: 10.1016/j.str.2013.08.005. View

12.

Vranken W, Boucher W, Stevens T, Fogh R, Pajon A, Llinas M . The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins. 2005; 59(4):687-96. DOI: 10.1002/prot.20449. View

13.

Belato H, East K, Lisi G . H, C, N backbone and side chain resonance assignment of the HNH nuclease from Streptococcus pyogenes CRISPR-Cas9. Biomol NMR Assign. 2019; 13(2):367-370. PMC: 6831365. DOI: 10.1007/s12104-019-09907-9. View

14.

Pritisanac I, Degiacomi M, Alderson T, Carneiro M, Ab E, Siegal G . Automatic Assignment of Methyl-NMR Spectra of Supramolecular Machines Using Graph Theory. J Am Chem Soc. 2017; 139(28):9523-9533. DOI: 10.1021/jacs.6b11358. View

15.

DAstolfo D, Pagliero R, Pras A, Karthaus W, Clevers H, Prasad V . Efficient intracellular delivery of native proteins. Cell. 2015; 161(3):674-690. DOI: 10.1016/j.cell.2015.03.028. View

16.

Leaver-Fay A, Tyka M, Lewis S, Lange O, Thompson J, Jacak R . ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol. 2010; 487:545-74. PMC: 4083816. DOI: 10.1016/B978-0-12-381270-4.00019-6. View

17.

Monneau Y, Rossi P, Bhaumik A, Huang C, Jiang Y, Saleh T . Automatic methyl assignment in large proteins by the MAGIC algorithm. J Biomol NMR. 2017; 69(4):215-227. PMC: 5764113. DOI: 10.1007/s10858-017-0149-y. View

18.

Wright A, Sternberg S, Taylor D, Staahl B, Bardales J, Kornfeld J . Rational design of a split-Cas9 enzyme complex. Proc Natl Acad Sci U S A. 2015; 112(10):2984-9. PMC: 4364227. DOI: 10.1073/pnas.1501698112. View

19.

Ruschak A, Kay L . Methyl groups as probes of supra-molecular structure, dynamics and function. J Biomol NMR. 2009; 46(1):75-87. DOI: 10.1007/s10858-009-9376-1. View

20.

Sprangers R, Kay L . Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature. 2007; 445(7128):618-22. DOI: 10.1038/nature05512. View