Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein’s functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMRstructures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism

Complex lasso : new entangled motifs in proteins

We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules

The Unique Cysteine Knot Regulates the Pleotropic Hormone Leptin

Leptin plays a key role in regulating energy intake/expenditure, metabolism and hypertension. It folds into a four-helix bundle that binds to the extracellular receptor to initiate signaling. Our work on leptin revealed a hidden complexity in the formation of a previously un-described, cysteine-knotted topology in leptin. We hypothesized that this unique topology could offer new mechanisms in regulating the protein activity. A combination of in silico simulation and in vitro experiments was used to probe the role of the knotted topology introduced by the disulphide-bridge on leptin folding and function. Our results surprisingly show that the free energy landscape is conserved between knotted and unknotted protein, however the additional complexity added by the knot formation is structurally important. Native state analyses led to the discovery that the disulphide-bond plays an important role in receptor binding and thus mediate biological activity by local motions on distal receptor-binding sites, far removed from the disulphide-bridge. Thus, the disulphide-bridge appears to function as a point of tension that allows dissipation of stress at a distance in leptin

Complex lasso: new entangled motifs in proteins

We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules

Novel knotted structure

We discovered hidden complexity in the cysteine-knotted topology of the cytokine leptin characterized by a covalent loop (a so-called zero knot) where part of a terminus is slipknotted through the zero knot. We call this motif a Cysteine Knotted Helical Bundle (CKHB). Up to date, there have been no reports of four-helix bundles with similar threaded topology. We explored the question: Do other proteins contain similar CKHBs? We discovered 11 proteins with similar threaded topology. However, leptin is the only motif with a C-terminal zero knot whereas all other structures have an N-terminal zero knot.Structure-based models were used to in investigate the folding/threading mechanism for six four-helix bundles: four with a threaded topology and two unknotted cytokine homologs. We found that the order of events in folding of the four-helix-bundle is conserved and that the nucleation site for folding is the C-terminal helix. Leptin uses a variation of the same mechanism, but in a unique reversed order in which large structural components of the protein start out as part of the zero knot. This contrasts with the other four-helix bundles, which use large structural components as a scaffold for loop formation. Remarkably, leptin slipknots large structure parts through the C-terminal zero knot while the other CKHBs threads its C-terminal through the N- terminal zero knot like a thread through a needle (a so-called plugging mechanism). Conclusively, since four-helix bundles have similar functional and folding landscape it is important to point out that CHKBs with an N-terminal loop pin down the N-terminal helix (helix A), while leptin has the opposite zero knot, thus keeping the N-terminal dynamic. Crystal structures and modelling of receptor complexes reveals one conserved interface (helix A interacting with the receptor) within the cytokines. This suggests a more dynamic assembly process between leptin and its receptor where the malleability of helix A could affect binding affinity and signaling.Non UBCUnreviewedAuthor affiliation: UCSD, CTBP (The Center for Theoretical Biological Physics)Postdoctora

Uncovering the Molecular Details Behind Disease-Associated Leptin Mutations and the Role of the Pierced Lasso Topology

Non UBCUnreviewedAuthor affiliation: Rice UniversityPostdoctora

Folding of the Ribosomal protein S6 : The role of sequence connectivity, overlapping foldons, and parallel pathways

To investigate how protein folding is affected by sequence connectivity five topological variants of the ribosomal protein S6 were constructed through circular permutation.  In these constructs, the chain connectivity (i.e. the order of secondary-structure elements) is changed without changing the native-state topology.  The effects of the permutations on the folding process were then characterised by φ-value analysis, which estimates the extent of contact formations in the transition-state ensemble.  The results show that the folding nuclei of the wild-type and permutant proteins comprises a common motif of one α-helix docking against two β-sheets, i.e. the minimal structure for folding.  However, this motif is recruited in different parts of the S6 structure depending on the permutation, either in the α1 or α2 half of the protein.  This minimal structure is not unique for S6 but can also be seen in other proteins.  As an effect of the dual nucleation possibilities, the transition-state changes describe a competition between two parallel pathways, which both include the central β-stand 1.  This strand constitutes thus a structural overlap between the two competing nuclei.  As similar overlap between competing nuclei is also seen in other proteins, I hypothesise that the coupling of several small nuclei into extended ‘super nuclei’ represents a general principle for propagating folding cooperativity across large structural distances.  Moreover, I demonstrate by NMR analysis that the existence of multiple folding nuclei renders the H/D-exchange kinetics independent of the folding pathway.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper IV: Manuscrip