Exposing Hidden Alternative Backbone Conformations in X-ray Crystallography Using qFit.

Area of Science:

• Structural Biology
• Computational Biology
• Biochemistry

Background:

• Proteins function by transitioning between conformational states, but crystal structures represent a time-averaged snapshot.
• Identifying alternative backbone conformations in electron density maps is challenging due to overlapping atomic positions.
• Understanding protein conformational heterogeneity is crucial for elucidating molecular mechanisms and drug design.

Purpose of the Study:

• To enhance the qFit program for modeling protein conformational heterogeneity from X-ray diffraction data.
• To incorporate peptide flips and alternative glycine conformations into structural modeling.
• To investigate the role of these conformational changes in protein function and inhibitor design.

Main Methods:

• Utilized mixed integer quadratic programming (MIQP) in the qFit program to explore numerous sidechain and backbone combinations.
• Developed and implemented major modeling enhancements for peptide flips and alternative glycine conformations.
• Applied the enhanced qFit model to analyze X-ray diffraction data, including HIV protease structures.

Main Results:

• Identified four stereotypical clusters of peptide flips, frequently occurring at glycine residues (n+1 position).
• Observed distinct peptide flip patterns in HIV protease associated with different inhibitor binding.
• Demonstrated that peptide flips, often facilitated by glycine flexibility, act as conformational switches causing significant local rearrangements.

Conclusions:

• Enhanced modeling of backbone heterogeneity with high-resolution X-ray data provides critical insights into protein dynamics.
• Peptide flips and glycine flexibility are important conformational elements that can be computationally modeled.
• This approach advances the structure-function relationship understanding and aids in designing novel therapeutic inhibitors.