We have found from our ST simulations that N17Htt
forms two primary states: a single extended helix and a two-helix bundle of which the c-terminal helix exhibits some degree of flexibility. Both states display a large hydrophobic face opposite a region of alternating charge in amphipathic fashion. Tam et. al10
have previously predicted N17Htt
to form an amphipathic alpha helix as a result of experimental data and a sequence comparison search. We have found the system can adopt multiple stable thermodynamic states, of which the dominant state at relevant temperatures is a two-helix bundle. This two-helix bundle state initially seems inconsistent with experimental findings, but in fact it agrees well with experimental data. With respect to the experimental mutation studies, insertion of two Proline “helix-breaker” residues within residues L4
was shown to completely halt aggregation, indicating that this region is highly helical. In our computational study, those same residues are found to adhere almost exclusively to this regime, showing near 90% helicity at 300K mid stretch. Since the two helical regions are separated by Ala10
, our proposed two-helix structure is not in contrast to the experimental findings.
Our structures agree very well with the main notion that N17Htt
is amphipathic. While the polar residues and the hydrophobic face are the driving factors for the two-helix conformation, they appear to compete for the precise configuration the two helices will adopt with relation to one another. Although in some structural clusters we see the most prominent feature as a salt-bridge network, in the structure characterized as the ensemble's free energy minimum, the two helices create a large uninterrupted hydrophobic face. Such a conformation would favor hydrophobic packing through dimerization to reduce its surface area. This also adheres to Tam et. al
's previous work10
in which they found the hydrophobic residues as a prerequisite for aggregation, and the polar residues to significantly control the rate and extent of aggregation.
It has been shown that Htt aggregation is nucleation dependent.14
This infers the existence of a critical transition, which has been hypothesized to be monomeric beta formation in the polyQ tail. It has also been shown that the rate limiting step in the aggregation pathway involves N17Htt 10
. Our simulation results provide two possible mechanisms for such a nucleation event.
It is shown that N17Htt has binding interactions with both itself and the polyQ domains. It is energetically favorable for N17Htt to dimerize in such a way as to pack the two hydrophobic faces together, simultaneously exposing the charged residues to solvent while minimizing non-polar surface area. In order for intra-chain N17Htt-polyQ binding interactions to exist, a turn is required to have been formed by N17Htt or PolyQ. A hydrogen bonding network between the charged residues of N17Htt and the polar polyQ side chains would contribute to these binding interactions by creating a beta-like strand. Moreover, the correct formation of this beta-like strand might be crucial for the nucleation event. Both mechanisms we propose are based on the formation of a beta-strand structure, which we feel is the key role N17Htt plays in Htt aggregation kinetics. However, they differ in whether N17Htt or PolyQ makes the turn for the initial beta-strand.
In the first mechanism, the turn connecting two-helix bundle in N17Htt will naturally serve as the turn. We have observed a degree of flexibility in the C-terminal helix orientation and conformation, (alpha-helix, 3-10-helix, disordered, etc.) The correct C-terminal helix orientation could help to facilitate the polyQ repeat domain interactions with N17Htt by allowing the chain to more easily wrap back on itself and keeping both domains in close proximity. (See )
Fig 9 Cartoons showing the N17Htt-PolQ structures in two proposed mechanisms described in the text. Two representative cluster centroid structures are shown with faces paired to minimize exposed hydrophobic surface area. Each shows a model polyQ tail (cyan) (more ...)
In the second mechanism, the polyQ chain makes the turn and N17Htt
adopts a single helix conformation in the aggregates. In this case, there would be an additional step. An initial transition is needed for N17Htt
from its dominant two-helix bundle state into the single helix state. Subsequently, the polyQ tail forms the turn for the beta-strand like configuration. This has the potential of being a rare event, and also of being able to propagate similar events in a nucleating fashion due to the lengthened scaffold of charged residues it presents. In addition, this mechanism puts more emphasis on the polar residue layout, and less on a covalent tether between the two domains. This could prove insightful due to an observed increase in aggregation kinetics with the addition of unbound N17Htt
A general topology coinciding with these two structures was proposed by Tam et al10
. Moreover, the concept of N17Htt
as a scaffold fits in nicely with the current hypotheses regarding Htt fibril structure and rate-limiting nucleation events. The end-to-end length of the N17Htt
polar region corresponds roughly to one turn of the current model for β-sheet elongation, excluding the turn residues. Wetzel et. al.
performed a series of experimental point mutation studies in which induced β-turns interspersed by 9-10 glutamine residues showed aggregation potential nearly as efficient as polyGln4515
. In contrast, peptides containing 7-8 glutamines between β-turns aggregated much less readily, indicating periodic β turns staggered every 9-10 glutamines in the aggregate form. In addition, we have found previous computational studies of polyGln to indicate a repeating β-turn topology of similar length17
. We feel these matching dimensions strongly support the N17Htt
role as a molecular scaffold.
There are several current ideas representing the rate-limiting nucleation step. Since it is generally accepted that monomeric polyglutamine exists as a random coil, a globular to β-sheet transition is required. There are currently two acknowledged possibilities capable of explaining the free-energetic pathway for this transition8
, both of which would benefit from the idea of N17Htt
acting as a molecular scaffold.
Chen et al.8
propose the single chain critical nucleus to be a “compact β-sheet” of roughly four turns and of very high β content. Such a structure would be a local free energy minimum or meta-stable state, having each of the four segments stabilized through hydrogen bonds with the neighboring intra-chain segment. The problem arising in reaching such a conformation is that initially there are no existing β-segments for an un-coiled stretch to interact with. N17Htt
would facilitate this process by presenting a constant stretch of accessible polar residues for hydrogen bonding by an uncoiled region of glutamines.
Crick et al.18
point out the possibility of the critical nucleus existing as a free energy maximum in the case of a single structure, or a heterogeneous mix of high energy structures. In this case the stability would come only after fibrillar addition through interchain interactions. This is similar to the first transition, but differs by existing as a local free energy maximum needing inter-chain interactions to adopt its configuration permanently. Pre-fibril, two or more of these events might be required simultaneously and in close spatial proximity to induce fibril formation. N17Htt
would again present a constant source of polar residues, reducing or eliminating the need for simultaneous globule to rod-like transitions by acting as a source of interchain interactions. In addition, any hydrophobic induced dimerization caused by N17Htt
can be imagined to be quite beneficial with this type of transition as well, due to its dependence on the proximity of other chains.