Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

Gauto, Diego F.; Macek, Pavel; Malinverni, Duccio; Fraga, Hugo; Paloni, Matteo; Sučec, Iva; Hessel, Audrey; Bustamante, Juan Pablo; Barducci, Alessandro; Schanda, Paul

doi:10.1038/s41467-022-29423-0

Cited by 18 publications

(19 citation statements)

References 90 publications

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“…REDOR and NERRD measurements on specifically methyl-labeled samples showed that the loop samples a wide conformational space within the cavity and is able to stabilize ligands in the active site, whereby a conserved histidine plays a key role. The data provided strong evidence for the functional importance of highly dynamic parts of enzymes and the potential of MAS NMR to investigate their dynamics at atomic resolution …”

Section: Structural and Dynamical Biology By 1h-detected Mas Nmrmentioning

confidence: 83%

“…The data provided strong evidence for the functional importance of highly dynamic parts of enzymes and the potential of MAS NMR to investigate their dynamics at atomic resolution. 361 Using the same model system, Schanda and co-workers demonstrated that 1 H-detected fast MAS NMR combined with a tailored isotope labeling scheme can provide insight into central functional and structural role of aromatic residues. A particular difficulty in observing their resonances in large proteins is dynamics and limited spectral dispersion.…”

Section: H-based Dynamicsmentioning

confidence: 99%

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¹H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

et al. 2022

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Since the first pioneering studies on small deuterated peptides dating more than 20 years ago, 1H detection has evolved into the most efficient approach for investigation of biomolecular structure, dynamics, and interactions by solid-state NMR. The development of faster and faster magic-angle spinning (MAS) rates (up to 150 kHz today) at ultrahigh magnetic fields has triggered a real revolution in the field. This new spinning regime reduces the 1H–1H dipolar couplings, so that a direct detection of 1H signals, for long impossible without proton dilution, has become possible at high resolution. The switch from the traditional MAS NMR approaches with 13C and 15N detection to 1H boosts the signal by more than an order of magnitude, accelerating the site-specific analysis and opening the way to more complex immobilized biological systems of higher molecular weight and available in limited amounts. This paper reviews the concepts underlying this recent leap forward in sensitivity and resolution, presents a detailed description of the experimental aspects of acquisition of multidimensional correlation spectra with fast MAS, and summarizes the most successful strategies for the assignment of the resonances and for the elucidation of protein structure and conformational dynamics. It finally outlines the many examples where 1H-detected MAS NMR has contributed to the detailed characterization of a variety of crystalline and noncrystalline biomolecular targets involved in biological processes ranging from catalysis through drug binding, viral infectivity, amyloid fibril formation, to transport across lipid membranes.

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Section: Structural and Dynamical Biology By 1h-detected Mas Nmrmentioning

confidence: 83%

Section: H-based Dynamicsmentioning

confidence: 99%

¹H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

et al. 2022

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“…However, the interconversion between these two states happens on a much faster timescale than any other protein activity (49). Schanda et al found that the enzymatic activity of the TET2 aminopeptidase is controlled by the conformational equilibrium of a highly dynamic loop in the catalytic chamber (50). Also, the turnover in the HisFH enzyme complex is dictated by the population of the active state, with the conversion between ground and active state taking place on a faster time scale than enzymatic activity (51).…”

Section: Discussionmentioning

confidence: 99%

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Scheerer

Adkar

Bhattacharyya

et al. 2022

Preprint

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Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. Mutation of multiple conserved residues leads to increased substrate inhibition, suggesting a positive selection during evolution. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

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“…However, modeling dynamics this way beyond a two-state exchange is challenging, due to experimental limitations and poor timescale separation. In effect, we are limited to highly simplified models of the complex underlying dynamics of our data where the structure of states often remain elusive or ambiguous [23][24][25][26][27][28].…”

Section: Introductionmentioning

confidence: 99%

Rescuing off-equilibrium simulation data through dynamic experimental data with dynAMMo

Kolloff,

Olsson

2023

Mach. Learn.: Sci. Technol.

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Long-timescale behavior of proteins is fundamental to many biological processes. Molecular dynamics (MD) simulations and biophysical experiments are often used to study protein dynamics. However, high computational demands of MD limit what timescales are feasible to study, often missing rare events, which are critical to explain experiments. On the other hand, experiments are limited by low resolution. We present dynamic augmented Markov models (dynAMMo) to bridge the gap between these data and overcome their respective limitations. For the first time, dynAMMo enables the construction of mechanistic models of slow exchange processes that have been not observed in MD data by integrating dynamic experimental observables. As a consequence, dynAMMo allows us to bypass costly and extensive simulations, yet providing mechanistic insights of the system. Validated with controlled model systems and a well-studied protein, dynAMMo offers a new approach to quantitatively model protein dynamics on long timescales in an unprecedented manner.

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Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

Cited by 18 publications

References 90 publications

¹H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

¹H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Rescuing off-equilibrium simulation data through dynamic experimental data with dynAMMo

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Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

Cited by 18 publications

References 90 publications

1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Rescuing off-equilibrium simulation data through dynamic experimental data with dynAMMo

Contact Info

Product

Resources

About

¹H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

¹H-Detected Biomolecular NMR under Fast Magic-Angle Spinning