Abstract
Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-β-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleus marisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.
| Original language | English |
|---|---|
| Pages (from-to) | 1846-1856 |
| Number of pages | 11 |
| Journal | Biophysical Journal |
| Volume | 123 |
| Issue number | 13 |
| DOIs | |
| State | Published - Jul 2 2024 |
Funding
We acknowledge funding to R.L.L. from NSF MCB grant 1817796 and Georgia Institute of Technology COVID relief funds, to G.M.T. from the ACS Bridge Program and GAANN grant P200A210014 , to the Idea Proteomics facility at AUMS from NIH grant R24GM137786 , and to J.C.G. from NIH grant R01GM148586 . We acknowledge the core facilities at the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology for the use of their shared equipment, services, and expertise. This work benefited from the use of the SasView application, originally developed under NSF Award DMR-0520547 . SasView contains code developed with funding from the European Union’s Horizon 2020 research and innovation program under the SINE2020 Project , grant agreement no. 654000 . Neutron scattering studies at the CG-3 Bio-SANS instrument at the High-Flux Isotope Reactor operated by the Oak Ridge National Laboratory were sponsored by the Office of Biological and Environmental Research and by the Scientific User Facilities Division , Office of Basic Energy Sciences , U.S. Department of Energy . This manuscript has been coauthored by UT-Battelle, LLC, under contract no. DE-AC05- 401 00OR22725 with the U.S. Department of Energy . The United States Government retains, and the publisher, by accepting the article for publication, acknowledges that the United States 404 Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ). We acknowledge funding to R.L.L. from NSF MCB grant 1817796 and Georgia Institute of Technology COVID relief funds, to G.M.T. from the ACS Bridge Program and GAANN grant P200A210014, and to J.C.G. from NIH grant R01GM148586. We acknowledge the core facilities at the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology for the use of their shared equipment, services, and expertise. This work benefited from the use of the SasView application, originally developed under NSF Award DMR-0520547. SasView contains code developed with funding from the European Union's Horizon 2020 research and innovation program under the SINE2020 Project, grant agreement no. 654000. Neutron scattering studies at the CG-3 Bio-SANS instrument at the High-Flux Isotope Reactor operated by the Oak Ridge National Laboratory were sponsored by the Office of Biological and Environmental Research and by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. This manuscript has been coauthored by UT-Battelle, LLC, under contract no. DE-AC05- 401 00OR22725 with the U.S. Department of Energy. The United States Government retains, and the publisher, by accepting the article for publication, acknowledges that the United States 404 Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). The authors have no financial or other interests to disclose. This manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ).