Hosur, R. V. | bioRxiv Science

bioRxiv2022

Intrinsic disorder in CENP-ACse4 tail and its chaperone facilitates synergistic association for kinetochore stabilization

The kinetochore is a complex multiprotein network that assembles at a specialized DNA locus called the centromere to ensure faithful chromosome segregation. The centromere is epigenetically marked by a histone H3 variant - the CenH3. The budding yeast CenH3, called Cse4, consists of an unusually long and disordered N-terminal tail that has a role in kinetochore assembly. Its disordered chaperone, Scm3 is involved in its centromeric deposition as well as in the maintenance of a segregation-competent kinetochore. The dynamics of the Cse4 N-tail and chaperone interaction have not been studied, leaving a gap in our understanding of their roles at the centromere. Previously, we had shown that Scm3 is an intrinsically disordered protein. Here, using NMR and a variety of biophysical and bioinformatics tools, we show that Cse4 N-tail is also disordered, the two proteins interact with each other at multiple sites, and this interaction reduces the disorder in Scm3; the chain opens up relative to the native state ensemble and the conformational exchange is reduced. Interestingly, this interaction between the two intrinsically disordered protein is fairly specific as seen by positive and negative controls, and is majorly driven by electrostatics as both the proteins have multiple acidic and basic regions. The complex retains a fair amount of disorder, which facilitates a synergistic association with the essential inner kinetochore Ctf19-Mcm21-Okp1-Ame1 complex; a model has been suggested to this effect. Given the abundance of intrinsic disorder in the kinetochore proteins, this type of interaction and adaptation may be prevalent in other proteins as well for mediating kinetochore assembly. Thus, the present study, on one hand, provides significant structural and mechanistic insights into the complex and dynamic process of kinetochore assembly, and on the other hand, illustrates a mechanism that intrinsically disordered proteins would adapt to mediate the formation of complex multiprotein networks, in general. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/504061v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8fe5cforg.highwire.dtl.DTLVardef@138afb1org.highwire.dtl.DTLVardef@96943forg.highwire.dtl.DTLVardef@1deb673_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2018

Mechanistic insights from replica exchange molecular dynamics simulations into mutation induced disordered-to-ordered transition in Hahellin, a βγ-crystallin

Intrinsically disordered proteins (IDPs) form a special category because they lack a unique well-folded 3D structure under physiological conditions. They play crucial role in cell signaling, regulatory functions and responsible for several diseases. Although, they are abundant in nature, only a small fraction of it has been characterized till date. Such proteins adopt a range of conformations and can undergo transformation from disordered-to-ordered state or vice-versa upon binding to ligand. Insights of such conformational transition is perplexing in several cases. In the present study, we characterized disordered as well as ordered states and the factors contributing the transitions through a mutational study by employing replica exchange molecular dynamics simulation on a {beta}{gamma}-crystallin. Most of the proteins within this superfamily are inherently ordered. However, Hahellin, although a member of {beta}{gamma}-crystallin, it is intrinsically disordered in its apo-form which takes a well-ordered {beta}{gamma}-crystallin fold upon binding to Ca2+. It is intriguing that the mutation at the 5th position of the canonical motif to Arg increases the domain stability in several ordered microbial {beta}{gamma}-crystallins with concomitant loss in Ca2+ binding affinity. We carried out similar Ser to Arg mutations at 5th position of the canonical motif for the first time in an intrinsically disordered protein to understand the mechanistic insights of conformational transition. Our study revealed that newly formed ionic and hydrogen bonding interactions at the canonical Ca2+ binding sites play crucial role in transforming the disordered conformation into ordered {beta}{gamma}-crystallin.\n\nAuthor summaryIntrinsically disordered proteins lack a unique ordered 3D structure under physiological condition. Although, they are abundant in nature, only a small fraction of these proteins has been characterized till date due to adaptation of multiple conformations and methodological limitation. {beta}{gamma}-crystallins are inherently ordered, however recently a small number of proteins within this superfamily have been identified as intrinsically disordered protein. Hahellin is one such protein which is intrinsically disordered in its apo-form but takes a well-ordered {beta}{gamma}-crystallin fold upon binding to Ca2+. In the present study, we decipher the underlying mechanism of disordered-to-ordered transition in Hahellin by mutations, employing replica exchange molecular dynamics simulations. Earlier experimental studies reported an increase in stabilization of the ordered {beta}{gamma}-crystallion upon mutation to Arg at 5th position of the canonical Ca2+ binding motifs, N/D-N/D-X1-X2-S/T-S. We performed similar Ser to Arg mutation in an intrinsically disordered Hahellin to get the mechanistic insights of the conformational transition in the absence of Ca2+. Our study revealed that several newly formed ionic and hydrogen bonding interactions contributed by the mutant residues are responsible for both intra- and inter-motif rigidification, resulting in overall stability of {beta}{gamma}-crystallin domain.

biophysics↗