Abstract

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ∼200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.

Highlights

  • Crystallization and Data Collection—Two microliters of selenomethionine-substituted Sec7d were mixed in with 2 ␮l of mother liquor containing 15% (w/v) PEG 1000 and 0.1 M sodium citrate, pH 5.0

  • The HR5562A14.13 plasmid was transformed into codon-enhanced BL21(DE3) pMGK Escherichia coli and cultured in MJ9 minimal medium containing selenomethionine, lysine, phenylalanine, threonine, isoleucine, leucine, and valine for SeMet labeling as described [11]

  • Expressed proteins were purified using an A KTAxpressTM (GE Healthcare) two-step protocol consisting of HisTrap HP affinity chromatography followed directly by HiLoad 26/60 Superdex 75 gel filtration chromatography

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Summary

Introduction

Crystallization and Data Collection—Two microliters of selenomethionine-substituted Sec7d were mixed in with 2 ␮l of mother liquor containing 15% (w/v) PEG 1000 and 0.1 M sodium citrate, pH 5.0. Crystals were grown at 18 °C by the hanging drop vapor diffusion method, cryoprotected with 20% (v/v) glycerol, and flash frozen in liquid nitrogen. Several diffraction data sets were collected at the beamlines X4A, X4C, and X6A of the National Synchrotron Light Source at Brookhaven National Laboratory. Data were processed with the HKL2000 package [12]. The crystals belong to P32 space group with unit cell parameters a ϭ b ϭ 53.9 Å and c ϭ 75.7 Å. There is one monomer per asymmetric unit. Residues beyond Ala-828 are disordered in the current structure

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