Structural and Thermodynamic Characterization of Nore1-SARAH: A Small, Helical Module Important in Signal Transduction Networks

Abstract

Tumor suppressor Nore1, its acronym coming from novel Ras effector, is one of the 10 members of the Rassf (Ras association domain family) protein family that have been identified. It is expressed as two mRNA splice variants, Nore1A and a shorter isoform, Nore1B. It forms homo- and heterocomplexes through its C-terminal SARAH (Sav/Rassf/Hpo) domain. The oligomeric state of Nore1 and other SARAH domain-containing proteins is important for their cellular activities. However, there are few experimental data addressing the structural and biophysical characterization of these domains. In this study, we show that the recombinant SARAH domain of Nore1 crystallizes as an antiparallel homodimer with representative characteristics of coiled coils. As is typical for coiled coils, the SARAH domain shows a heptad register, yet the heptad register is interrupted by two stutters. The comparisons of the heptad register of Nore1-SARAH with the primary structure of Rassf1-4, Rassf6, MST1, MST2, and WW45 indicate that these proteins have a heptad register interrupted by two stutters, too. Moreover, on the basis of the structure of Nore1-SARAH, we also generate structural models for Rassf1 and Rassf3. These models indicate that Rassf1- and Rassf3-SARAH form structures very similar to that of Nore1-SARAH. In addition, we show that, as we have previously found for MST1, the SARAH domain of Nore1 undergoes association-dependent folding. Nevertheless, the Nore1 homodimer has a lower affinity and thermodynamic stability than the MST1 homodimer, while the monomer is slightly more stable. Our experimental results along with our theoretical considerations indicate that the SARAH domain is merely a dimerization domain and that the differences between the individual sequences lead to different stabilities and affinities that might have an important functional role.

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

“…10D and are in the same range as previously published values: C. Herrmann and colleagues394041 determined a dissociation constant K D in the order of hundreds of nM for the RASSF5-MST1 complex in FRET experiments using stopped-flow fluorimetry, while the self-association constant for RASSF5 was found to be 5–10 μM, and that for MST1 was in the low nM range. In their case, K D was calculated as the ratio between the association rate constant k on and the dissociation rate constant k off .…”

H-ras Inhibits the Hippo Pathway by Promoting Mst1/Mst2 Heterodimerization

“…Modification with the thiol-reactive IAEDANS (Methods) yielded the AEDANS-labeled derivatives Mst1-AED, Mst2-AED and Mst2-LW-AED. Since SARAH domains associate in an antiparallel orientation [9, 10], a unique Trp residue at the N-terminal end of the Mst1 SARAH domain can be used as a FRET donor while the AEDANS label at the C-terminus of the adjacent subunit in the dimer serves as an acceptor (inset Figure 2F). For Mst2, an equivalent Trp residue was introduced by site-directed mutagenesis (Mst2-LW).…”

Structural dissection of Hippo signaling

“…The SARAH domain-mediated homodimerization of MST1/2 is required for their full activation [51]. The monomeric state of MST1/2 SARAH domain is thermodynamically unstable and its dimerization has been coupled with structural folding [49,52]. The structures of MST1/2 and RASSF5 SARAH homodimers or heterodimers have been determined (Fig.…”

“…The structures of MST1/2 and RASSF5 SARAH homodimers or heterodimers have been determined (Fig. 1B) [44,48,[52][53][54]. The SARAH domain of MST1/2 or RASSF5 alone forms a symmetric homodimer (PDB codes: 2JO8, 2YMY, 4HKD, 4L0N, 4NR2, and 4OH9).…”

“…Although the overall structures of these SARAH domains are similar, their local conformations vary, even in different structures of the same SARAH domain, implying a dynamic nature of the SARAH domain. Sequence conservation indicates that these structural features exist in all SARAH domains [52,54]. SAV1 binding enhances MST1/2 kinase activity, while the influence of RASSFs on MST1/2 activity is uncertain [41][42][43]51,55,56].…”