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LRAT-specific domain facilitates vitamin A metabolism by domain swapping in HRASLS3

Nature Chemical Biology volume 11, pages 26–32 (2015)Cite this article

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Abstract

Cellular uptake of vitamin A, production of visual chromophore and triglyceride homeostasis in adipocytes depend on two representatives of the vertebrate N1pC/P60 protein family, lecithin:retinol acyltransferase (LRAT) and HRAS-like tumor suppressor 3 (HRASLS3). Both proteins function as lipid-metabolizing enzymes but differ in their substrate preferences and dominant catalytic activity. The mechanism of this catalytic diversity is not understood. Here, by using a gain-of-function approach, we identified a specific sequence responsible for the substrate specificity of N1pC/P60 proteins. A 2.2-Å crystal structure of the HRASLS3–LRAT chimeric enzyme in a thioester catalytic intermediate state revealed a major structural rearrangement accompanied by three-dimensional domain swapping dimerization not observed in native HRASLS proteins. Structural changes affecting the active site environment contributed to slower hydrolysis of the catalytic intermediate, supporting efficient acyl transfer. These findings reveal structural adaptation that facilitates selective catalysis and mechanism responsible for diverse substrate specificity within the LRAT-like enzyme family.

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Figure 1: Design of HRASLS–LRAT chimeric proteins.
Figure 2: HRASLS–LRAT chimeras catalyze formation of retinyl esters.
Figure 3: Crystal structure of the HRASLS3–LRAT chimeric protein.
Figure 4: Effect of the structural rearrangement on the enzymatic activity.
Figure 5: The acylated form of HRASLS3–LRAT.
Figure 6: Phospholipid membrane topology of the HRASL3–LRAT chimera.

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Acknowledgements

We thank X. Tu and B.M. Kevany for initial X-ray diffraction data collection of HRASLS3–LRAT crystals and L.T. Webster, Jr. for help in preparation of this manuscript. This work was supported by grants EY023948 (M.G.) and EY009339 (K.P.) from the National Eye Institute of the National Institutes of Health (NIH), the Medical Scientist Training Program grant T32 GM007250 (A.E.S.) from the National Institutes of Health (NIH) as well as the Nutrition and Obesity Research Center at CWRU (M.G.). We thank the Northeastern Collaborative Access Team staff for assistance with diffraction data collection. Use of the Advanced Photon Source, an Office of the Science User Facility operated for the US Department of Energy Office of Science by the Argonne National Laboratory, was supported by the US Department of Energy under contract DE-AC02-06CH11357. Preliminary data for this study were obtained at beamline X29 of the National Synchrotron Light Source. Its financial support is derived principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the United States Department of Energy and by NIH Grant P41RR012408 from the US National Center for Research Resources and P41GM103473 from the US National Institute of General Medical Sciences. K.P. is John H. Hord Professor of Pharmacology.

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  1. Department of Pharmacology, Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA

    Marcin Golczak, Avery E Sears, Philip D Kiser & Krzysztof Palczewski

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  1. Marcin Golczak
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  3. Philip D Kiser
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Contributions

M.G. and K.P. designed the experiments. M.G. and A.E.S. performed biochemical experiments and crystallized HRASLS3–LRAT. M.G. and P.D.K. collected and processed the crystallographic data. All authors contributed to the data analyses. M.G. and A.E.S. wrote the manuscript with valuable input from P.D.K. and K.P.

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Correspondence to Marcin Golczak or Krzysztof Palczewski.

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Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–12. (PDF 2207 kb)

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Structural organization of the active site in HRASLS3–LRAT chimeric enzyme (MOV 14695 kb)

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Golczak, M., Sears, A., Kiser, P. et al. LRAT-specific domain facilitates vitamin A metabolism by domain swapping in HRASLS3. Nat Chem Biol 11, 26–32 (2015). https://doi.org/10.1038/nchembio.1687

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