RESUMEN
Formation of protein knots is an intriguing offshoot of the protein folding problem. Since experimental resolution on knot formation is limited, theoretical methods currently provide the most detailed insights into the knotting process. While suitable for shallow knots, molecular dynamics simulations have faced challenges capturing the formation of deep knots in proteins such as the minimally tied trefoil α/ß methyltransferase from Thermotoga maritima (MTTTM). To improve the efficiency of MTTTM knotting in Cα Go-model simulations, mutant variants of the MTTTM Go-model were investigated. Through a structure-based analysis of knotted and unknotted states, four residues (K71, R72, E75, V76) were identified to increase the knotting efficiency from 2% to 83% when their contact energies were doubled and dihedral strength around the knot loop increased. The key features of this model are (i) a C-terminal slipknot intermediate that threads the knot in a highly unstructured intermediate, (ii) the inability to knot in native-like intermediate states, and (iii) a minor population in a long-lived trap that cannot knot. Examination of residue 71-76 contacts provides a small set of potential mutants that can directly test the model's validity. In addition, the knotting optimization process developed here has broad applicability in generating knotting-efficient models of other knotted proteins.
RESUMEN
The methyltransferases that belong to the SpoU-TrmD family contain trefoil knots in their backbone fold. Recent structural dynamic and binding analyses of both free and bound homologs indicate that the knot within the polypeptide backbone plays a significant role in the biological activity of the molecule. The knot loops form the S-adenosyl-methionine (SAM)-binding pocket as well as participate in SAM binding and catalysis. Knots contain both at once a stable core as well as moving parts that modulate long-range motions. Here, we sought to understand allosteric effects modulated by the knotted topology. Uncovering the residues that contribute to these changes and the functional aspects of these protein motions are essential to understanding the interplay between the knot, activation of the methyltransferase, and the implications in RNA interactions. The question we sought to address is as follows: How does the knot, which constricts the backbone as well as forms the SAM-binding pocket with its three distinctive loops, affect the binding mechanism? Using a minimally tied trefoil protein as the framework for understanding the structure-function roles, we offer an unprecedented view of the conformational mechanics of the knot and its relationship to the activation of the ligand molecule. Focusing on the biophysical characterization of the knot region by NMR spectroscopy, we identify the SAM-binding region and observe changes in the dynamics of the loops that form the knot. Importantly, we also observe long-range allosteric changes in flanking helices consistent with winding/unwinding in helical propensity as the knot tightens to secure the SAM cofactor.
Asunto(s)
Metiltransferasas/química , Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Sitio Alostérico , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios Proteicos , Pliegue de ProteínaRESUMEN
Entanglement and knots occur across all aspects of the physical world. Despite the common belief that knots are too complicated for incorporation into proteins, knots have been identified in the native fold of a growing number of proteins. The discovery of proteins with this unique backbone characteristic has challenged the preconceptions about the complexity of biological structures, as well as current folding theories. Given the intricacies of the knotted geometry, the interplay between a protein's fold, structure, and function is of particular interest. Interestingly, for most of these proteins, the knotted region appears critical both in folding and function, although full understanding of these contributions is still incomplete. Here, we experimentally reveal the impact of the knot on the landscape, the origin of the bistable nature of the knotted protein, and broaden the view of knot formation as uniquely decoupled from folding.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Pliegue de Proteína , Modelos Moleculares , Estructura Secundaria de Proteína , Termodinámica , Thermotoga maritima/enzimologíaRESUMEN
Hysteresis is a signature for a bistability in the native landscape of a protein with significant transition state barriers for the interconversion of stable species. Large global stability, as in GFP, contributes to the observation of this rare hysteretic phenomenon in folding. The signature for such behavior is non-coincidence in the unfolding and refolding transitions, despite waiting significantly longer than the time necessary for complete denaturation. Our work indicates that hysteresis in the knotted protein, the minimal tied trefoil from Thermotoga maritma (MTTTm), is mediated by a network of side chain interactions within a tightly packed core. These initially identified interactions include proline 62 from a tight ß-like turn, phenylalanine 65 at the beginning of the knotting loop, and histidine 114 that initiates the threading element. It is this tightly packed region and the knotting element that we propose is disrupted with prolonged incubation in the denatured state, and is involved in the observed hysteresis. Interestingly, the disruption is not linked to backbone interactions, but rather to the packing of side chains in this critical region.
Asunto(s)
Proteínas Bacterianas/química , Conformación Proteica , Pliegue de Proteína , Thermotoga maritima/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , TermodinámicaRESUMEN
Deletion of the ß-bulge trigger-loop results in both a switch in the preferred folding route, from the functional loop packing folding route to barrel closure, as well as conversion of the agonist activity of IL-1ß into antagonist activity. Conversely, circular permutations of IL-1ß conserve the functional folding route as well as the agonist activity. These two extremes in the folding-functional interplay beg the question of whether mutations in IL-1ß would result in changes in the populations of heterogeneous folding routes and the signaling activity. A series of topologically equivalent water-mediated ß-strand bridging interactions within the pseudosymmetric ß-trefoil fold of IL-1ß highlight the backbone water interactions that stabilize the secondary and tertiary structure of the protein. Additionally, conserved aromatic residues lining the central cavity appear to be essential for both stability and folding. Here, we probe these protein backbone-water molecule and side chain-side chain interactions and the role they play in the folding mechanism of this geometrically stressed molecule. We used folding simulations with structure-based models, as well as a series of folding kinetic experiments to examine the effects of the F42W core mutation on the folding landscape of IL-1ß. This mutation alters water-mediated backbone interactions essential for maintaining the trefoil fold. Our results clearly indicate that this perturbation in the primary structure alters a structural water interaction and consequently modulates the population of folding routes accessed during folding and signaling activity.
Asunto(s)
Interleucina-1beta/química , Modelos Moleculares , Pliegue de Proteína , Sustitución de Aminoácidos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mutación , Fenómenos Ópticos , Conformación Proteica , Termodinámica , Agua/químicaRESUMEN
Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap?
RESUMEN
The pleiotropic pro-inflammatory cytokine interleukin (IL)-1ß has co-evolved with a competitive inhibitor, IL-1 receptor antagonist (IL-1Ra). IL-1ß initiates cell signaling by binding the IL-1 receptor (IL-1R) whereas IL-1Ra acts as an antagonist, blocking receptor signaling. The current paradigm for agonist/antagonist functions for these two proteins is based on the receptor-ligand interaction observed in the crystal structures of the receptor-ligand complexes. While IL-1Ra and IL-1ß are structurally homologous, IL-1Ra engages only two of the three extracellular domains of the receptor, whereas IL-1ß engages all three. We find that an allosteric functional switch exists within a highly conserved pocket of residues, residues 111-120. This region is maintained across all IL-1 family members and serves as a hydrophobic mini-core for IL-1ß folding. A key difference across species is a conserved aromatic residue at position 117 in IL-1ß, versus a conserved cysteine in IL-1Ra at the analogous position, 116. We find that the replacement of C116 with a phenylalanine switches the protein from an antagonist to an agonist despite the distant location of C116 relative to receptor interaction sites. These results suggest new ways to develop designer cytokine activity into the ß-trefoil fold and may be of general use in regulation of this large family of signaling proteins.
Asunto(s)
Regulación Alostérica , Proteína Antagonista del Receptor de Interleucina 1/química , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Mutación Puntual , Proteína Antagonista del Receptor de Interleucina 1/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación ProteicaRESUMEN
Interleukin-1ß (IL-1ß) is the cytokine crucial to inflammatory and immune response. Two dominant routes are populated in the folding to native structure. These distinct routes are a result of the competition between early packing of the functional loops versus closure of the ß-barrel to achieve efficient folding and have been observed both experimentally and computationally. Kinetic experiments on the WT protein established that the dominant route is characterized by early packing of geometrically frustrated functional loops. However, deletion of one of the functional loops, the ß-bulge, switches the dominant route to an alternative, yet, as accessible, route, where the termini necessary for barrel closure form first. Here, we explore the effect of circular permutation of the WT sequence on the observed folding landscape with a combination of kinetic and thermodynamic experiments. Our experiments show that while the rate of formation of permutant protein is always slower than that observed for the WT sequence, the region of initial nucleation for all permutants is similar to that observed for the WT protein and occurs within a similar timescale. That is, even permutants with significant sequence rearrangement in which the functional-nucleus is placed at opposing ends of the polypeptide chain, fold by the dominant WT "functional loop-packing route", despite the entropic cost of having to fold the N- and C- termini early. Taken together, our results indicate that the early packing of the functional loops dominates the folding landscape in active proteins, and, despite the entropic penalty of coalescing the termini early, these proteins will populate an entropically unfavorable route in order to conserve function. More generally, circular permutation can elucidate the influence of local energetic stabilization of functional regions within a protein, where topological complexity creates a mismatch between energetics and topology in active proteins.
Asunto(s)
Interleucina-1beta/química , Interleucina-1beta/metabolismo , Humanos , Interleucina-1beta/genética , Pliegue de Proteína , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
Proteins fold into three-dimensional structures in a funneled energy landscape. This landscape is also used for functional activity. Frustration in this landscape can arise from the competing evolutionary pressures of biological function and reliable folding. Thus, the ensemble of partially folded states can populate multiple routes on this journey to the native state. Although protein folding kinetics experiments have shown the presence of such routes for several proteins, there has been sparse information about the structural diversity of these routes. In addition, why a given protein populates a particular route more often than another protein of similar structure and sequence is not clear. Whereas multiple routes are observed in theoretical studies on the folding of interleukin-1ß (IL-1ß), experimental results indicate one dominant route where the central portion of the protein folds first, and is then followed by closure of the barrel in this ß-trefoil fold. Here we show, using a combination of computation and experiment, that the presence of functionally important regions like the ß-bulge in the signaling protein IL-1ß strongly influences the choice of folding routes. By deleting the ß-bulge, we directly observe the presence of route-switching. This route-switching provides a direct link between route selection and the folding and functional landscapes of a protein.
Asunto(s)
Interleucina-1beta/fisiología , Interleucina-1beta/química , Modelos Teóricos , Pliegue de ProteínaRESUMEN
Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (â¼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.
Asunto(s)
Ferredoxinas/química , Modelos Moleculares , Pliegue de Proteína , Regulación Alostérica/fisiología , Secuencias de Aminoácidos , Ferredoxinas/genética , Ferredoxinas/metabolismo , Humanos , Hierro/química , Hierro/metabolismo , Mutación , Estabilidad Proteica , Estructura Terciaria de Proteína , Azufre/química , Azufre/metabolismoRESUMEN
Interleukin-1beta (IL-1beta) is a cytokine within the beta-trefoil family. Our data indicate that the folding/unfolding routes are geometrically frustrated. Follow-up theoretical studies predicted backtracking events that could contribute to the broad transition barrier and the experimentally observed long-lived intermediate. The backtracking route is attributed to the topological frustration introduced by the packing of the functional loop (the beta-bulge, residues 47-53) to the nascent barrel. We used real-time refolding NMR experiments to test for the presence of backtracking events predicted from our theoretical studies. Structural variants of IL-1beta, a beta-bulge deletion, and a circular permutation that opens the protein in the middle of the experimentally observed kinetic intermediate, were also refolded and studied to determine the affects on the observed folding reactions. The functional loop deletion variant demonstrated less backtracking than in WT protein whereas the permutation still maintains backtracking in agreement with theoretical predictions. Taken together, these findings indicate that the backtracking results from geometric frustration introduced into the fold for functional purposes.