Abstract
Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis. Its long C-terminal proline-rich domain contains 13 PXXP motifs, which orchestrate its interactions with multiple proteins. The SH3 domains of syndapin and endophilin bind the PXXP motifs called Site 2 and 3 (Pro-786-Pro-793) at the N-terminal end of the proline-rich domain, whereas the amphiphysin SH3 binds Site 9 (Pro-833-Pro-836) toward the C-terminal end. In some proteins, SH3/peptide interactions also involve short distance elements, which are 5-15 amino acid extensions flanking the central PXXP motif for high affinity binding. Here we found two previously unrecognized elements in the central and the C-terminal end of the dynamin proline-rich domain that account for a significant increase in syndapin binding affinity compared with a previously reported Site 2 and Site 3 PXXP peptide alone. The first new element (Gly-807-Gly-811) is short distance element on the C-terminal side of Site 2 PXXP, which might contact a groove identified under the RT loop of the SH3 domain. The second element (Arg-838-Pro-844) is located about 50 amino acids downstream of Site 2. These two elements provide additional specificity to the syndapin SH3 domain outside of the well described polyproline-binding groove. Thus, the dynamin/syndapin interaction is mediated via a network of multiple contacts outside the core PXXP motif over a previously unrecognized extended region of the proline-rich domain. To our knowledge this is the first example among known SH3 interactions to involve spatially separated and extended long-range elements that combine to provide a higher affinity interaction.
Highlights
Dynamin is a GTPase that mediates vesicle fission during synaptic vesicle endocytosis
The Site 2 PXXP Motif in Dynamin I Is the Core Binding Site for Syndapin I—Previous work showed that the binding of syndapin to dynamin I required two components in the region 772RRSPTSSPTPQRRAPAVPPARPGSR796 of dynamin (10)
To test the hypothesis that the proposed N-terminal short distance elements (SDEs) directly binds to the SH3 domain independently of the Site 2 core, a range of dynamin I proline-rich domain (PRD) truncations and point mutants was generated, each containing different regions of the PRD (Fig. 1A)
Summary
DNA Constructs and Protein Expression—The rat dynamin PRD-Ia (amino acids Asn-746 –Leu-864) was amplified by PCR from a green fluorescent protein-tagged dynamin construct and subcloned into pGEX6P-1 (GE Healthcare) as described previously (12). Affinities for the syndapin1⁄7PRD and syndapin1⁄7C2 interactions were calculated from titrations of the unlabeled syndapinSH3 domain into 15N-labeled C2 or PRD In the former case the changes in amide proton chemical shift of Arg-784, Ala-785, and Val-788 were separately plotted as a function of the concentration of syndapin-SH3, and the data were fitted to simple 1:1 Langmuir binding isotherms. The dissociation constant was taken as the average of the three derived values, and the error was estimated as 20% In the latter case, the exchange between free and bound 15N-labeled PRD is slower (the socalled intermediate exchange regime), and the peak positions are, not a simple population-weighted average of the chemical shifts of the free and bound forms. A structure-based sequence alignment was conducted using STRAP program for statistical analysis
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