Autism-associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses.

Michael Bucher,Fatemeh Hassani Nia,Marina Mikhaylova,Stephan Niebling,Eunjoon Kim,Dmitry Molodenskiy,Michael R Kreutz,Alla S Kostyukova,Hans-Jürgen Kreienkamp,Dmitri Svergun,Yuhao Han

doi:10.7554/elife.66165

Abstract

Members of the SH3- and ankyrin repeat (SHANK) protein family are considered as master scaffolds of the postsynaptic density of glutamatergic synapses. Several missense mutations within the canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons.

Highlights

De novo and inherited point mutations contribute to several neuropsychiatric disorders and are common in genes that are responsible for synaptic function (Gratten et al, 2013; Hammer et al, 2015; Penzes et al, 2011)
The SHANK3a isoform shows highest expression in the striatum and hippocampus and consists of five distinct domains plus an additional proline-rich cluster (Jiang & Ehlers, 2013; Wang, Xu, et al, 2014). These include the SH3- and ankyrin-rich repeat (SHANK)/ProSAP N-terminal (SPN) domain followed by the ankyrin repeats (ARR), a Src homology 3 (SH3) domain, the post-synaptic density (PSD)-95/DLG/ZO-1 (PDZ) domain and the C29 terminal sterile alpha motif (SAM)
In this study, using a wide range of biophysical and cellular approaches we show that the autism spectrum disorders (ASDs)-associated point mutations R12C and L68P affect different levels of protein structure

Summary

Introduction

De novo and inherited point mutations contribute to several neuropsychiatric disorders and are common in genes that are responsible for synaptic function (Gratten et al, 2013; Hammer et al, 2015; Penzes et al, 2011). The SHANK3a isoform shows highest expression in the striatum and hippocampus and consists of five distinct domains plus an additional proline-rich cluster (Jiang & Ehlers, 2013; Wang, Xu, et al, 2014). These include the SHANK/ProSAP N-terminal (SPN) domain followed by the ankyrin repeats (ARR), a Src homology 3 (SH3) domain, the PSD-95/DLG/ZO-1 (PDZ) domain and the C29 terminal sterile alpha motif (SAM). SHANK3 acts as “master organizer” of the PSD via multiple protein interactions and the resulting indirect association with ionotropic glutamate receptors (Jiang & Ehlers, 2013; Zeng et al, 2016; Zhang et al, 2005) and is crucial for synaptic structure and function (Grabrucker et al, 2011; Monteiro & Feng, 2017)

Methods

Results

Conclusion