Structural Insights into the Specific Binding of Huntingtin Proline-Rich Region with the SH3 and WW Domains

Abstract

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

Interaction with Polyglutamine-expanded Huntingtin Alters Cellular Distribution and RNA Processing of Huntingtin Yeast Two-hybrid Protein A (HYPA)

“…1A) (24). Our previous work identified that the first and third portions of Htt PRR specifically interact with the tandem WW domains (13). To better understand how the tandem WW domains of HYPA interact with the Htt PRR motif, we used GST pulldown experiments to characterize the interactions between N-terminal Htt and different GST-fused WW domains (Fig.…”

“…The proteins were purified by Ni 2ϩ -NTA affinity column followed by gel filtration chromatography. 15 N/ 13 C-Labeled proteins were prepared by using the M9 minimal medium containing 15 15 N/ 13 C-labeled 2WW-P67T protein in a phosphate buffer (10 mM, pH 6.0) was used for the structural determination by NMR as our previous study (13). All data acquisition was carried out at 25°C on a Varian INOVA 600 spectrometer.…”

“…Our previous work elucidated the specific binding of Htt-PRR with SH3 and WW domains (13). However, how the expanded polyQ tract influences the interaction of Htt with HYPA and consequently impacts on the cellular function of HYPA are largely unknown.…”

Structural basis for recognition of the third SH3 domain of full‐length R85 (R85FL)/ponsin by ataxin‐7

“…Atx7 PP2 and SH3C bind by isothermal titration calorimetry (View Interaction: 1,2,3,4,5,6,7,8,9,10,11,12) Atx7 binds to SH3C by pull down (View interaction) Atx7 100Q and SH3C colocalize by fluorescence microscopy (View interaction) SH3C and Atx7 bind by nuclear magnetic resonance (View interaction)…”

“…After purification, MTSL ((1-oxyl-2,2,5,5-tetramethyl pyrroline-3-methyl) methanethiosulfonate) (ALEXIS Biochemicals, San Diego) was incubated with Atx7_PP2 at 4°C overnight. Ten-fold excess of MTSL was used for the reaction and the excess MTSL was removed by desalting [11]. In the PRE experiment, after collecting a HSQC spectrum, dithiothreitol (DTT, 10 mM) was added to the mixture in the NMR tube and stayed at room temperature for 5 h to remove MTSL from the Atx7_PP2 peptide, and then another HSQC spectrum was acquired for reference [15].…”