Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance

Abstract

Proteins composed of multiple domains allow for structural heterogeneity and interdomain dynamics that may be vital for function. Intradomain structures and dynamics can influence interdomain conformations and vice versa. However, no established structure determination method is currently available that can probe the coupling of these motions. The protein Pin1 contains separate regulatory and catalytic domains that sample “extended” and “compact” states, and ligand binding changes this equilibrium. Ligand binding and interdomain distance have been shown to impact the activity of Pin1, suggesting interdomain allostery. In order to characterize the conformational equilibrium of Pin1, we describe a novel method to model the coupling between intra- and interdomain dynamics at atomic resolution using multistate ensembles. The method uses time-averaged nuclear magnetic resonance (NMR) restraints and double electron–electron resonance (DEER) data that resolve distance distributions. While the intradomain calculation is primarily driven by exact nuclear Overhauser enhancements (eNOEs), J couplings, and residual dipolar couplings (RDCs), the relative domain distribution is driven by paramagnetic relaxation enhancement (PREs), RDCs, interdomain NOEs, and DEER. Our data support a 70:30 population of the compact and extended states in apo Pin1. A multistate ensemble describes these conformations simultaneously, with distinct conformational differences located in the interdomain interface stabilizing the compact or extended states. We also describe correlated conformations between the catalytic site and interdomain interface that may explain allostery driven by interdomain contact.

A Specific pSer/Thr-Pro Motif Generates Interdomain Communication Bifurcations of Two Modes of Pin1 in Solution Nuclear Magnetic Resonance

“…37 However, the T29-A31-S32 fragment does not exhibit a small peak when only the WW domain is measured. 33 The results of Born 34 support the idea that those at the WW-binding site, the PPIase catalytic site, and the NOE heel at the interdomain interface were simulated to be consistent with a bistable ensemble rather than a single state. The temperature modulates the distance between the noninteracting double domains (between 279 and 303 K 33 ), and the distance between the major and minor peaks decreases with an increase in temperature at T29 and A31 located in the WW domain.…”

“…The results of Zhu’s team hold that the minor peak should belong to Pin1 in the compact state population when it binds to a peptide that facilitates the promotion of interdomain commutation on the slow time scale. However, the time-averaged NMR and DEER used by Born’s team suggest that the dominant component in the NMR is the compact state population (70%). Although the obtained minor peaks imply that the anchoring case of Zhu does not consider the minor peaks of F139-A140-L141, a certain population distribution is likely lost in the calculation of the anchored–separated state.…”

“…Because the compact state exhibited in X-ray is due to PEG400, , PEG400 acts as a small molecule binder stabilizing the conformation of both structural domains. In addition, Born’s team in 2021 described the existence of multiple conformational modes of Pin1 in solution NMR . The dynamic equilibrium of Pin1, comprising the compact and extended states, is also recorded in the two-dimensional HSQC (heteronuclear single-quantum coherence) spectra; this dynamic transition occurs on the fast time scale of NMR.…”

“…In addition, Born's team in 2021 described the existence of multiple conformational modes of Pin1 in solution NMR. 34 The dynamic equilibrium of Pin1, comprising the compact and extended states, is also recorded in the two-dimensional HSQC (heteronuclear single-quantum coherence) spectra; 35 this dynamic transition occurs on the fast time scale of NMR. Zhu's team has suggested the existence of a 29:71 ratio of the compact state by the build anchored substrate and extended state by only the PPIase domain, 31 whereas Born's team suggests a 70:30 34 ratio between the compact and extended states by measuring the distance of the relative domain distribution.…”

The effect of spin polarization on double electron–electron resonance (DEER) spectroscopy

“…Double electron-electron resonance (DEER) spectroscopy is a pulse electron paramagnetic resonance (EPR) technique utilized for determining distances between spin centers on a nanometer scale (Milov et al, 1981;Larsen and Singel, 1993;Milov and Tsvetkov, 1997). The technique has seen use with studies of organic polymers but is most commonly used for structural studies in large biomolecules, such as proteins and nucleic acids (Schiemann and Prisner, 2007;Tavenor et al, 2014;Duss et al, 2014;Manglik et al, 2015;Verhalen et al, 2017;Barth et al, 2018;Dastvan et al, 2019;Evans et al, 2020;Born et al, 2021). DEER resolves the full distribution of distances in an ensemble of proteins, making it possible to directly quantify protein conformational ensembles (Larsen and Singel, 1993;Jeschke and Polyhach, 2007;Jeschke, 2012).…”