The cryo-electron microscopy structure of huntingtin

Abstract

SummaryHuntingtin (Htt) is a large (348 kDa) protein, essential for embryonic development and involved in diverse cellular activities such as vesicular transport, endocytosis, autophagy and transcription regulation1,2. While an integrative understanding of Htt's biological functions is lacking, the large number of identified interactors suggests that Htt serves as a protein-protein interaction hub1,3,4. Furthermore, Huntington’s disease is caused by a mutation in the Htt gene, resulting in a pathogenic expansion of a polyglutamine (polyQ) repeat at the N-terminus of Htt5,6. However, only limited structural information on Htt is currently available. Here we employed cryo-electron microscopy (cryo-EM) to determine the structure of full-length human Htt in a complex with HAP40/F8A7 to 4 Å resolution. Htt is largely α-helical and consists of three major domains. The N- and C-terminal domains contain multiple HEAT repeats arranged in a solenoid fashion. These domains are connected by a smaller bridge domain containing different types of tandem repeats. HAP40 is also largely α-helical and has a tetratricopeptide repeat (TPR)-like organization. HAP40 binds in a cleft contacting the three Htt domains by hydrophobic and electrostatic interactions, thereby stabilizing Htt conformation. These data rationalize previous biochemical results and pave the way for an improved understanding of Htt’s diverse cellular functions.

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

“…Purification of the HTT-HAP40 complex, previously reported from an adherent mammalian cell expression system (23), could be achieved through a 3:1 viral titre ratio of HTT 1-3144 Q23 in a C-terminally FLAG-tagged pBMDEL expression construct and HAP40 in an N-terminally His6tagged pFBOH-MHL insect cell expression vector ( Figure 2C). The purification protocol was modified so that an additional Ni-affinity chromatography step was included following the FLAG pull-down.…”

“…Size-exclusion chromatography of HTT protein derived from insect Sf9 or mammalian EXPI293F cells using a Superose6 10/300 GL column gives a characteristic elution profile (Figure 2A), with a void-aggregate peak followed by peaks previously attributed as being dimer and monomer species of HTT based on column standards (14)(15)(16). The recent cryo-electron microscopy structure of HTT in complex with HAP40 reveals a bi-lobed structure of HTT in which the N-HEAT and C-HEAT domains wrap around HAP40 yielding a more compact and globular structure (23). Furthermore, HAP40 was described as being critical for producing a conformationally homogenous HTT sample amenable to cryoelectron microscopy structure determination.…”

Piecing together the puzzle of Huntington’s disease

“…An analysis of the structure of huntingtin 4 published in March supports the idea that it is some kind of molecular chaperone -a protein that aids the function of other proteins -says Kochanek, who led the work. It took a decade to come to fruition, he adds, because the protein's extreme flexibility made it difficult for researchers to get clear images of the structure.…”

“…The number of CAG repeats in HTT varies among species of vertebrate, and their expansion is greatest in humans. Huntingtin is essential for the development of the nervous system before birth; indeed, the researchers contend that CAG repeats might have contributed to the development of the complexity of the vertebrate brain 4 . But other researchers say that CAG repeats are just as likely to have arisen by chance, and that they have little effect until a threshold is crossed.…”

Evolutionary Analyses of RNA Editing and Amino Acid Recoding in Cephalopods

“…In humans, expansions of the polyglutamine domain in HTT lead to Huntington's Disease, a progressive neurodegenerative disorder ( 36 ). HTT is also widely expressed throughout human development and is predicted to be highly pleiotropic; however, much is not known about its normal functions (36)(37)(38). Investigation of HTT protein diversity in cephalopods could provide a unique perspective on the function and structure of this protein.…”