Simulation Validation Against Experimental Data

This repository contains the methodology and results for validating MD simulations using HDX-MS experimental data. The validation was conducted using the Python package HDXer and the BestVendruscolo method to compute protection factors for the residues.

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To compute protection factors for the residues, the following predictive model was used:

$$\ln P_i = (\beta_C N_{C,i} + \beta_H N_{H,i})$$

HDXer: Backbone Amide Protection Factor Estimation

This model estimates 𝑃𝑖 (protection factor) for each backbone amide as an ensemble average function of:

• Heavy atom contacts
• H bonds formed by the amide NH group.

The number of H bonds and heavy atom contacts formed by each amide NH group determines the protection factor for each backbone amide.

By default, HDXer evaluates H bonds and contacts similarly to the originally-parameterized model:

• H bonds are counted if there is a protein oxygen atom within 2.4 Å of the amide H atom.
• Heavy-atom contacts are computed as the total number of nearby protein non-hydrogen atoms within 6.5 Å of the amide N atom.
• Contacts from sequence neighbors of the amide (residues i-2 to i+2) are excluded from the total.

This phenomenological model includes two empirical scaling parameters to reflect the relative contributions of:

• Heavy atom contacts (𝛽𝐶)
• H bonding (𝛽𝐻)

For this study, the default values were used:

• 𝛽𝐶 = 0.35
• 𝛽𝐻 = 2.0

These values were parameterized using a training dataset of globular, monomeric proteins.

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Using the calculated protection factors, HDXer computes the fractional deuteration of each amide at a nominal deuterium exposure time (𝐷𝑖,𝑡) using:

$$𝐷𝑖,𝑡 = 1 - \exp(-k_{int,i} t / P_i)$$

where:

• 𝑘int,𝑖 = Intrinsic rate of exchange for each amide.
  • Automatically computed by HDXer based on experimentally-determined reference data.

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Experimental Conditions

The HDX-MS experiments were conducted under the following conditions:

• Temperature: 293 K
• pD: 7.9

These parameters were incorporated into the predictive model.

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Computational and Experimental Comparison

Three independent replicas for the following systems were analyzed:

1. Full-length PI3Kα WT
2. ΔABD p110α WT

The H/D exchange rates were predicted for all available fragments at:

• 3 seconds
• 30 seconds
• 300 seconds

The computational results were then compared to experimental data.

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References

1. P.S. Lee, R.T. Bradshaw, F. Marinelli, K. Kihn, A. Smith, P.L. Wintrode, et al. Interpreting hydrogen-deuterium exchange experiments with molecular simulations: tutorials and applications of the HDXer ensemble reweighting software. Living J Comput Mol Sci (2021); 3: 1521. DOI: 10.33011/livecoms.3.1.1521

2. R.T. Bradshaw, F. Marinelli, J.D. Faraldo-Gómez, L.R. Forrest Interpretation of HDX Data by Maximum-Entropy Reweighting of Simulated Structural Ensembles. Biophys J (2020); 118: 1649-1664. DOI: 10.1016/j.bpj.2020.02.005

3. R.B. Best, M. Vendruscolo Structural interpretation of hydrogen exchange protection factors in proteins: characterization of the native state fluctuations of CI2. Structure (2006); 14: 97-106. DOI: 10.1016/j.str.2005.09.012

4. Y. Bai, J.S. Milne, L. Mayne, S.W. Englander Primary structure effects on peptide group hydrogen exchange. Proteins (1993); 17: 75-86. DOI: 10.1002/prot.340170110

5. D. Nguyen, L. Mayne, M.C. Phillips, S.W. Englander Reference parameters for protein hydrogen exchange rates. J Am Soc Mass Spectrom (2018); 29: 1936-1939. DOI: 10.1007/s13361-018-2021-z