Backbone-independent NMR resonance assignments of methyl probes in large proteins

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.

Enhanced Specificity Mutations Perturb Allosteric Signaling in CRISPR-Cas9

“…11 Indeed, due to the size of its polypeptide chain (i.e., 160 kDa), the Cas9 protein challenges traditional solution NMR, requiring optimized constructs of the individual domains to report on its structural and dynamical features. 11,28 To address the allosteric mechanism, it is however critical to characterize the communication network within the full-length Cas9. Toward this aim, we performed all-atom MD simulations of the CRISPR-Cas9 system in its wild-type (WT) form 9 and introducing the K848A, K810A and K855A mutations.…”

“…Finally, it is worth noting that the combination of solution NMR and molecular simulations enabled us to characterize the allosteric signaling from the individual HNH domain with experimental accuracy, to the full-length Cas9 with atomic level detail through MD simulations. This “bottom-up” approach exploits the capability of solution NMR to identify allosteric motions within optimized constructs of the multi-domain Cas9 protein, 11,28 while all-atom MD simulations are used to characterize the communication network within the full-length Cas9. Future studies in our laboratories will leverage this approach to fully characterize the allosteric transmission across the multiple domains of Cas9 and its variants, gaining thorough insights on the system’s function and specificity.…”

Progress toward automated methyl assignments for methyl-TROSY applications

“…The models can be derived from crystallography, homology (Bordoli et al, 2009), comparative (Song et al, 2013), or artificial intelligence modeling (Baek et al, 2021;Jumper et al, 2021). Computational optimization of side chain rotamers in experimentally derived models improves the agreement with the solution-state data and the accuracy of the resulting assignments (Nerli et al, 2021). Care must be taken to select a methyl labeling scheme that produces the most unique LNs without compromising data quality.…”

“…There are currently six NOE-based approaches available: MAP-XSII (Xu and Matthews, 2013), FLAMEnGO2.0 (Chao et al, 2014), MAGMA (Pritisanac et al, 2017), MAGIC (Monneau et al, 2017), Methyl-FLYA (Pritisanac et al, 2019), and MAUS (Nerli et al, 2021). Limited performance and accuracy comparisons have been conducted previously (Nerli et al, 2021;Pritisanac et al, 2020).…”

“…There are currently six NOE-based approaches available: MAP-XSII (Xu and Matthews, 2013), FLAMEnGO2.0 (Chao et al, 2014), MAGMA (Pritisanac et al, 2017), MAGIC (Monneau et al, 2017), Methyl-FLYA (Pritisanac et al, 2019), and MAUS (Nerli et al, 2021). Limited performance and accuracy comparisons have been conducted previously (Nerli et al, 2021;Pritisanac et al, 2020). Differences in input requirement and the lack of standard dataset for indiscriminate use across all software platforms make unbiased evaluation difficult and true blind tests or meta-analysis of the results arduous (Table 1).…”