Investigating the Molecular Basis of Oligomerisation Specificity Using Coiled Coil Peptides - Essay Example

The Effects of Mutation on Leucine Zipper Coiled Coil Structure Introduction Currently, coiled coils are successfully and extensively used in the designing of multi-stranded structures that are applied in biotechnology, basic research, nanotechnology, and medicine and material science. The broad application range and the critical functions played by this structures in about all biological processes indicates the need for understanding the factors that oligomerization and coiled coil folding in details. Some studies have addressed the unresolved and important questions related to the why presence of specific oligomerization-state determinants that is within coiled coil is not linked with its topology (Steinmetz et al., 2007). The studies have identified that there is unexpected and general correlation between trigger sequences (Indispensable elements for coiled coil formation) and coiled coil oligomerization state specificity .Through the use yeast archytype coiled-coil domain transcriptional factor activator GCN4 as a model system, it was found that oligomerization state determinants that are trimer specific and well established ,switched the topology of the peptide to a trimer from a dimer, only after being inserted into the trigger sequence. The results were confirmed with 2 other coiled coil dimmers that is not related (cortexillin-1 and ATF1).It is further indicated that multiple topology determinants have the capacity of co-existing in the same trigger sequence, and this reveals a delicate balance of the resultant oligomerization state through forces that are position dependent (Steinmetz et al., 2007). Due to the simplicity of this leucine zipper, the alpha helical coiled coil has been traditionally used in a broad number of studies that have targeted the key principles of protein stability, oligomerization and folding (Steinmetz et al., 2007).In this case, the coiled coils currently are exploited as multipurpose tool in various applications seen to be increasing steadily as well as ranging from basic research to medicine. The relationship between specific oligomerization and topology It is well known that what is referred to as trigger sequence plays a critical role in controlling coiled –coil formation. The distinct feature of several trigger sequences is that they have a capacity of folding into several reasonable stable monomeric helices before the formation of the coiled coil structure (Kammerer et al.1998) Recently, the NMR structures of peptide spanning the GCN4 trigger sequence was solved and indicated that the structure is stabilized by electrostatic interactions and a network of hydrogen bonds (Steinmetz et al., 2007) The trigger sequence helical conformation is seen to provide a local structural scaffold that is effective for major residues interaction of the chains that are colliding. Folding and competent helices do form a nucleation site that enhances the productivity in chain association. There is then a zip up of the interacting helices along the molecule in order to direct the formation of a stable structure of coiled –coil .Various trigger sequence proteins show considerable diversity, and this indicates the impossibility of the existence of the consensus sequences (Steinmetz et al., 2007).Consequently, the trigger sequences prediction on the basis of sequence information alone is still a big challenge and needs experimental verification It is well known that specific oligomerization that is state determinant doesn’t relatively correlate with the corresponding topology of coiled coil. Despite the R-hxxhE, trimerization motif is abundantly found in several diverse protein families that harbor 3 stranded coiled coil domains, it is as well observed in some anti-parallel trimmers and dimmers like the transcriptional activators ATF1 and GCN4 (Schumacher et al.,2000).In order to be able to know the molecular basis of such discrepancy, a detailed structural and biophysical study was carried out to address how topology of the two stranded coiled coil model system can be impacted in cases where a given oligomerization state determinant are positioned at various coiled coil positions. In this case, the fundamental and unresolved questions on the way position specific motifs that is in the coiled coil defines its topology was addressed. According to the results, the far-UV CD spectra that were recorded from the 3 mutants and GCN4-p1 at 5oC were observed to be proteins with high alpha helix content. The peptide stabilities were evaluated through thermal unfolding profiles that was recorded by CD at 222nm.It was observed that all peptides had a sigmoid-shaped unfolding and this was consistent with the native like coiled coil structures. GCN4-pM2 and GCN4-pM1 sedimentation equilibrium analysis indicated that they were dimmers with similar or lower stability respectively, as compared to the wild type. In contrast, GCN4-pM3 had to yield an average molecular mass that is consistent with the trimeric structure formation despite the existence of the Asn16 at a heptad repeat at position a and this indicated a substantially higher melting temperature as compared to GCN4p1.In this case, the introduced mutation into this given GCN4-p1 variant is seen to fall into trigger sequence (Zaccai et al.,2011). Mutation impacts on coiled coil assembly and folding The most earliest and scrupulous studied alpha helical coiled coil sequences of proteins is known to be yeast transcriptional factor GCN4, dimerization domain. The 33-residue GCN4 leucine zipper normally known as GCN4-p1 is seen to be canonical coiled coil sequence that has a Leu and Val residues which are more pronounced at positions of d and a heptad respectively. Asn residue, single polar in nature, and located at a central a position is known to have a key role in regulating folding by forming a contact that is inter-chain polar with the hydrophobic dimer core (Akey et al., 2001) .For at least 2 decades since it was first described, the leucine-zipper domain, GCN4-p1 has acted as the basis for various critical studies on the folding of alpha helical coiled-coil. Despite, mutations have been linked to oligomerization state alteration (Gonzalez et al., 1996), the sequence of wild-type basically has been assumed to specify a dimer exclusively. According to some literature, it was indicated that mutations that occurs in GCN4-p1 at positions of a/d heptad are known to modify the preferred oligomerization sequence state (Ciani et al.,2010) .For instance, a mutant that had Ile at core positions of all a as well as Leu at all core positions of d (GCN4-pIL) were identified to form a dimer, in the due course, the switching to Ile at all positions of d (GCN4-pLI) and Leu at all positions of a resulted to a tetramer. The most interesting observation was that the Leu at d, Val at a native sequence hydrophobic core was seen to be not properly discriminated between trimer and dimer in the situation where the single core of Asn was absent. The leucine zipper wild-type GCN4 structure is known to have been determined previously. In this case, the leucine zipper is parallel coiled coil besides being two-stranded (Schnarr et al., 2002). Mutants of leucine zipper series, which occur as a result of residues’ mutations on the interface between the two leucine zipper’s helices, have been designed in order to understand the impact of such mutations on coiled coil assembly and folding, such as helix orientation and oligomeric state of the coiled coil(Ciani et al.,2010).There are other zippers that have been designed apart from this leaucine zipper, and they include a tryptophan zipper, a phenylalanine zipper and alanine zipper(Ciani et al.,2010).Mutants of GCN4 leucine zipper were designed in the previous studies based on a, d, e, g residues mutations that are around or on the interface between the two wide-type leucine zipper helices. Some are known to have been constructed by residues of non-interfacial mutation, and are known to be hydrophilic as well as are on the other helix face that is opposite to the interface that is between the 2 helices. In order to address the non-interfacial residues mutation impacts on coiled coil assembly and folding, that include the crossing angle, oligomeric state, and the anti-parallel or parallel coiled coil configuration, leucine zipper mutant was constructed with 4 hydrophilic non-interfacial residues mutations, K3A, D7A, Y17W, and H18N. The mutant of leucine zipper is seen to correspond to GCN4 C-terminus (249–281) of yeast and the numbering of the residues is from 1 - 33. The 4 mutations is observed to correspond to a heptad repeat at positions including b, f, b, c, in, respectively. In this case, the first three mutations ranging from hydrophilic residues to hydrophobic residues causes the non-interfacial helix face to be more hydrophobic. The results indicate a novel longitudinal leucine zipper association. This is a structural basis for viewing the longitudinal association of the protein fibers that are self-assembling. According to the results, it is indicated that the leucine zipper domain (GCN4-p1) has the capacity of adopting either a trimeric or dimeric coiled-coil fold, and this is based on the environment. Crystal structures with high-resolution indicates the way the residue of core Asn16 can be integrated into each state of oligomerization. Measurements of biophysical indicates populations of both assemblies of trimeric and dimeric in solution under a given conditions of the experiment. Simulations of Microsecond molecular dynamics (MD) gives details on the two folded states relative stabilities in isolation. The simulations involved parallel tempering application, and it is known to be a critical case of the replica exchange known to be as enhanced method of sampling. The identification of the peptides was confirmed by mass spectrometry with the use of a Voyager DE pro MALDI-TOF instrument that was calibrated with external standards (Ciani et al., 2010). References Akey DL, Malashkevich VN, Kim PS.(2001). Buried polar residues in coiled-coil interfaces. Biochemistry.;40:6352–6360 Ciani B, Bjelic S, Honnappa S, Jawhari H, Jaussi R, Payapilly A, Jowitt T, Steinmetz MO, Kammerer RA(2010). Molecular basis of coiled-coil oligomerization-state specificity. Proc. Natl. Acad. Sci. USA.;107:19850–19855. Gonzalez L, Brown RA, Richardson D, Alber T (1996). Crystal structures of a single coiled-coil peptide in two oligomeric states reveal the basis for structural polymorphism. Nat. Struct. Biol.;3:1002–1010. Kammerer RA,et al.(1998) An autonomous folding unit mediates the assembly of two-stranded coiled coils. Proc Natl Acad Sci USA 95:13419–13424. Schnarr NA, Kennan AJ(2002). Peptide tic-tac-toe: Heterotrimeric coiled-coil specificity from steric matching of multiple hydrophobic side chains. J. Am. Chem. Soc.;124:9779–9783 Schumacher MA, Goodman RH, Brennan RG(2000) The structure of a CREB bZIP.somatostatin CRE complex reveals the basis for selective dimerization and divalent cation-enhanced DNA binding. J Biol Chem 275:35242–35247. Steinmetz MO,et al.(2007) Molecular basis of coiled-coil formation. Proc Natl Acad Sci USA 104:7062–7067 Zaccai NR, Chi B, Thomson AR, Boyle AL, Bartlett GJ, Bruning M, Linden N, Sessions RB, Booth PJ, Brady RL, Woolfson DN (2011). A de novo peptide hexamer with a mutable channel. Nat. Chem. Biol.;7:935–941. Read More