Structure and membrane interaction of myristoylated ARF1

Abstract

ADP-ribosylation factors (ARFs) are small (21 kDa), monomeric GTPases that are important regulators of membrane traffic. When membrane bound, they recruit soluble adaptors to membranes and trigger the assembly of coating complexes involved in cargo selection and vesicular budding. N-myristoylation is a conserved feature of all ARF proteins that is required for its biological functions, although the mechanism(s) by which the myristate acts in ARF functions is not fully understood. Here we present the structure of a myristoylated ARF1 protein, determined by solution NMR methods, and an assessment of the influence of myristoylation on association of ARF1.GDP and ARF1.GTP with lipid bilayers. A model in which myristoylation contributes to both the regulation of guanine nucleotide exchange and stable membrane association is supported.

Figure 1

¹⁵N-¹H HSQC spectra of myr(+)- and myr(−) – yARF1·GDP (blue and red respectively). Blown-up views of chemical shift perturbed residues are displayed on the bottom.

Figure 2

Structure of myristoylated yARF1 and comparisons to related structures. a. Overlap of 15 accepted myr(+)-yARF1·GDP structures out of 100 trials. GDP is shown in blue and myristoyl in purple. b. The myristoyl binding pocket with leucines shown in red, isoleucines in blue, tyrosine in cyan and myristoyl in purple. c. The myristoyl binding pocket lies under the N-terminal amphiphilic helix in the myr(−)-hARF·GDP structure (PDB:1hur). d. The inter-strand loop λ3 clashes with the myristoyl chain in the GTP-bound conformation but not in the GDP-bound conformation. Backbone of myr(+)-yARF1·GDP is shown in ivory, myr(−)-hARF1·GDP in blue, hARF1Δ17·GTP in green. λ3 of all molecules is shown in red, and myristoyl is shown in purple.

Figure 3

a & b, The residue-specific effective rotational correlation times of myr(-) and myr(+)-yARF1·GDP in the absence or presence of 10% DMPC/DHPC small bicelles. c. One dimensional ¹⁵N-¹H HSQC scans of myr(−) and myr(+)-yARF1·GDP mixed with DMPC/DHPC bicelles of varying sizes. d. Effective rotational correlation times of myr(−) and myr(+)-yARF1·GTP in the presence of 10% DMPC/DHPC small bicelles.