Phase changes in T(3)R(3)(f) human insulin: temperature or pressure induced?

Area of Science:

• Biochemistry
• Structural Biology
• Crystallography

Background:

• Human insulin exists as hexamers crucial for storage and release.
• Understanding insulin's structural dynamics is key to diabetes research.
• Previous room-temperature structures provide a baseline for low-temperature analysis.

Purpose of the Study:

• To determine the high-resolution structure of T(3)R(3) hexameric human insulin at 100 K.
• To investigate conformational changes and zinc ion coordination at low temperatures.
• To compare the low-temperature structure with its room-temperature counterpart.

Main Methods:

• X-ray crystallography at 100 K using two different crystals.
• High-resolution data collection (1.2 and 1.3 Å resolution).
• Refinement of the crystal structures.

Main Results:

• Detailed structure of T(3)R(3) hexameric human insulin determined at 100 K.
• Observed rotation of insulin dimers and displacements in R(f)-state monomers.
• Identified four zinc ions on the threefold axis with varied coordination, including mixed tetrahedral/octahedral.
• Discovered novel zinc binding sites at dimer interfaces and alternate HisB10 conformations.
• Observed glycerol and water molecules in phenolic binding sites, alongside zinc ions and chloride in the second hexamer.
• Evidence of protonation for a GluB13 side chain due to specific molecular contacts.

Conclusions:

• The low-temperature structure reveals distinct dimer orientations and monomer displacements compared to room temperature.
• Novel zinc coordination environments and binding sites provide insights into insulin's stability and interactions.
• The findings contribute to a deeper understanding of human insulin's structural behavior under different thermal conditions.