Fly odorant-binding protein with high-histidine N-terminal extension binds to transition metals

Authors: SHAH, JAEE SHAILESH - University Of Texas At San Antonio; Buckmeier, Beverly - Greta; GRIFFITH, WENDELL - University Of Texas At San Antonio; Olafson, Pia; Perez De Leon, Adalberto - Beto; RENTHAL, ROBERT - University Of Texas At San Antonio

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/26/2021
Publication Date: 12/31/2021
Citation: Shah, J., Buckmeier, B.G., Griffith, W., Olafson, P.U., Perez De Leon, A.A., Renthal, R. 2021. Fly odorant-binding protein with high-histidine N-terminal extension binds to transition metals. Insect Biochemistry and Molecular Biology. 141. Article 103707. https://doi.org/10.1016/j.ibmb.2021.103707.
DOI: https://doi.org/10.1016/j.ibmb.2021.103707

Interpretive Summary: The role of odorant- and pheromone-binding proteins (OBPs) in olfactory function is not fully understood. We found an OBP sequence from the stable fly, Stomoxys calcitrans, ScalOBP60, that encodes a histidine- and acidic amino acid-rich segment at the start of the mature protein (N-terminal), suggesting a possible metal binding activity. A scan of the public sequence database revealed that these histidine-rich extensions were present in other fly OBPs as well as in beetle, wasp and ant OBPs but at the end of the mature protein in the latter. We produced the ScalOBP60 mature protein using a bacterial expression system and we also produced a truncated version of the protein that lacks the N-terminal histidine-rich region, tScalOBP60. Using mass spectrometry, we detected two different types of metal-binding sites. Copper, nickel and zinc bind to the histidine-rich region at the N-terminal, and copper further binds to an internal sequence position. Comparison of the spectra for ScalOBP60 versus tScalOBP60 showed that the histidine-rich sequence is structurally disordered, but it becomes more ordered in the presence of metal. When copper is bound to the internal site, binding of a hydrophobic ligand to ScalOBP60 is inhibited. The internal and N-terminal metal sites interact, possibly through a balance of conformations, suggesting a mechanism for metal regulation of ligand binding to ScalOBP60. Based on our studies of ScalOBP60, we propose several possible olfactory and non-olfactory functions for this OBP.

Technical Abstract: The role of odorant- and pheromone-binding proteins (OBPs) in olfactory function is not fully understood. We found an OBP sequence from the stable fly, Stomoxys calcitrans, ScalOBP60, that has a 25 amino acid N-terminal extension with a high content of histidine and acidic amino acids, suggesting a possible metal binding activity. A search of public databases revealed a large number of other fly OBPs with histidine-rich N-terminal extensions, as well as beetle, wasp and ant OBPs with histidine-rich C-terminal extensions. We recombinantly expressed ScalOBP60, as well as a truncated sequence which lacks the histidine-rich N-terminal region, tScalOBP60. Using fluorescence quenching and electrospray quadrupole time-of-flight mass spectrometry (ESI-QTOF), we detected two different types of metal-binding sites. Divalent copper, nickel and zinc binds to the N-terminal histidine-rich region, and divalent copper binds to an internal sequence position. Comparison of the ESI-QTOF spectra of ScalOBP60 and tScalOBP60 showed that the histidine-rich sequence is structurally disordered, but it becomes more ordered in the presence of divalent metal. When copper is bound to the internal site, binding of a hydrophobic ligand to ScalOBP60 is inhibited. The internal and N-terminal metal sites interact, possibly through a conformational equilibrium, suggesting a mechanism for metal regulation of ligand binding to ScalOBP60. Based on our studies of ScalOBP60, we propose several possible olfactory and non-olfactory functions for this OBP.