Is There a Functional Role for the Knotted Topology in Protein UCH-L1?

Knotted proteins are present in nature, but there is still an open issue regarding the existence of a universal role for these remarkable structures. To address this question, we used classical molecular dynamics (MD) simulations combined with in vitro experiments to investigate the role of the Gordian knot in the catalytic activity of UCH-L1. To create an unknotted form of UCH-L1, we modified its amino acid sequence by truncating several residues from its N-terminus. Remarkably, we find that deleting the first two N-terminal residues leads to a partial loss of enzyme activity with conservation of secondary structural content and knotted topological state. This happens because the integrity of the N-terminus is critical to ensure the correct alignment of the catalytic triad. However, the removal of five residues from the N-terminus, which significantly disrupts the native structure and the topological state, leads to a complete loss of enzymatic activity. Overall, our findings indicate that UCH-L1's catalytic activity depends critically on the integrity of the N-terminus and the secondary structure content, with the latter being strongly coupled with the knotted topological state.

Interfacial Dynamics and Growth Modes of ß2-Microglobulin Dimers.

Protein aggregation is a complex process, strongly dependent on environmental conditions and highly structurally heterogeneous, both at the final level of fibril structure and intermediate level of oligomerization. Since the first step in aggregation is the formation of a dimer, it is important to clarify how certain properties of the latter (e.g., stability or interface geometry) may play a role in self-association. Here, we report a simple model that represents the dimer's interfacial region by two angles and combine it with a simple computational method to investigate how modulations of the interfacial region occurring on the ns-µs time scale change the dimer's growth mode. To illustrate the proposed methodology, we consider 15 different dimer configurations of the ß2m D76N mutant protein equilibrated with long Molecular Dynamics simulations and identify which interfaces lead to limited and unlimited growth modes, having, therefore, different aggregation profiles. We found that despite the highly dynamic nature of the starting configurations, most polymeric growth modes tend to be conserved within the studied time scale. The proposed methodology performs remarkably well taking into consideration the nonspherical morphology of the ß2m dimers, which exhibit unstructured termini detached from the protein's core, and the relatively weak binding affinities of their interfaces, which are stabilized by nonspecific apolar interactions. The proposed methodology is general and can be applied to any protein for which a dimer structure has been experimentally determined or computationally predicted.

Effects of sequence-dependent non-native interactions in equilibrium and kinetic folding properties of knotted proteins.

Determining the role of non-native interactions in folding dynamics, kinetics, and mechanisms is a classic problem in protein folding. More recently, this question has witnessed a renewed interest in light of the hypothesis that knotted proteins require the assistance of non-native interactions to fold efficiently. Here, we conduct extensive equilibrium and kinetic Monte Carlo simulations of a simple off-lattice C-alpha model to explore the role of non-native interactions in the thermodynamics and kinetics of three proteins embedding a trefoil knot in their native structure. We find that equilibrium knotted conformations are stabilized by non-native interactions that are non-local, and proximal to native ones, thus enhancing them. Additionally, non-native interactions increase the knotting frequency at high temperatures, and in partially folded conformations below the transition temperatures. Although non-native interactions clearly enhance the efficiency of transition from an unfolded conformation to a partially folded knotted one, they are not required to efficiently fold a knotted protein. Indeed, a native-centric interaction potential drives the most efficient folding transition, provided that the simulation temperature is well below the transition temperature of the considered model system.

A Note on the Effects of Linear Topology Preservation in Monte Carlo Simulations of Knotted Proteins.

Monte Carlo simulations are a powerful technique and are widely used in different fields. When applied to complex molecular systems with long chains, such as those in synthetic polymers and proteins, they have the advantage of providing a fast and computationally efficient way to sample equilibrium ensembles and calculate thermodynamic and structural properties under desired conditions. Conformational Monte Carlo techniques employ a move set to perform the transitions in the simulation Markov chain. While accepted conformations must preserve the sequential bonding of the protein chain model and excluded volume among its units, the moves themselves may take the chain across itself. We call this a break in linear topology preservation. In this manuscript, we show, using simple protein models, that there is no difference in equilibrium properties calculated with a move set that preserves linear topology and one that does not. However, for complex structures, such as those of deeply knotted proteins, the preservation of linear topology provides correct equilibrium results but only after long relaxation. In any case, to analyze folding pathways, knotting mechanisms and folding kinetics, the preservation of linear topology may be an unavoidable requirement.

The folding space of proteinß2-microglobulin is modulated by a single disulfide bridge.

Protein beta-2-microglobulin (ß2m) is classically considered the causative agent of dialysis related amyloidosis, a conformational disorder that affects patients undergoing long-term hemodialysis. The wild type (WT) form, the ΔN6 structural variant, and the D76N mutant have been extensively used as model systems ofß2m aggregation. In all of them, the native structure is stabilized by a disulfide bridge between the sulphur atoms of the cysteine residues 25 (at B strand) and 80 (at F strand), which has been considered fundamental inß2m fibrillogenesis. Here, we use extensive discrete molecular dynamics simulations of a full atomistic structure-based model to explore the role of this disulfide bridge as a modulator of the folding space ofß2m. In particular, by considering different models for the disulfide bridge, we explore the thermodynamics of the folding transition, and the formation of intermediate states that may have the potential to trigger the aggregation cascade. Our results show that the dissulfide bridge affects folding transition and folding thermodynamics of the considered model systems, although to different extents. In particular, when the interaction between the sulphur atoms is stabilized relative to the other intramolecular interactions, or even locked (i.e. permanently established), the WT form populates an intermediate state featuring a well preserved core and two unstructured termini, which was previously detected only for the D76N mutant. The formation of this intermediate state may have important implications in our understanding ofß2m fibrillogenesis.

A tale of two tails: The importance of unstructured termini in the aggregation pathway of ß2-microglobulin.

The identification of intermediate states for folding and aggregation is important from a fundamental standpoint and for the design of novel therapeutic strategies targeted at conformational disorders. Protein human ß2-microglobulin (HB2m) is classically associated with dialysis-related amyloidosis, but the single point mutant D76N was recently identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. Here, we use D76N as a model system to explore the early stage of the aggregation mechanism of HB2m by means of an integrative approach framed on molecular simulations. Discrete molecular dynamics simulations of a structured-based model predict the existence of two intermediate states populating the folding landscape. The intermediate I1 features an unstructured C-terminus, while I2 , which is exclusively populated by the mutant, exhibits two unstructured termini. Docking simulations indicate that I2 is the key species for aggregation at acidic and physiological pH contributing to rationalize the higher amyloidogenic potential of D76N relative to the wild-type protein and the ΔN6 variant. The analysis carried out here recapitulates the importance of the DE-loop in HB2m self-association at a neutral pH and predicts a leading role of the C-terminus and the adjacent G-strand in the dimerization process under acidic conditions. The identification of aggregation hot-spots is in line with experimental results that support the importance of Phe56, Asp59, Trp60, Phe62, Tyr63, and Tyr66 in HB2m amyloidogenesis. We further predict the involvement of new residues such as Lys94 and Trp95 in the aggregation process.

Calcium binding to gatekeeper residues flanking aggregation-prone segments underlies non-fibrillar amyloid traits in superoxide dismutase 1 (SOD1).

Calcium deregulation is a central feature among neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Calcium accumulates in the spinal and brain stem motor neurons of ALS patients triggering multiple pathophysiological processes which have been recently shown to include direct effects on the aggregation cascade of superoxide dismutase 1 (SOD1). SOD1 is a Cu/Zn enzyme whose demetallated form is implicated in ALS protein deposits, contributing to toxic gain of function phenotypes. Here we undertake a combined experimental and computational study aimed at establishing the molecular details underlying the regulatory effects of Ca(2+) over SOD1 aggregation potential. Isothermal titration calorimetry indicates entropy driven low affinity association of Ca(2+) ions to apo SOD1, at pH7.5 and 37°C. Molecular dynamics simulations denote a noticeable loss of native structure upon Ca(2+) association that is especially prominent at the zinc-binding and electrostatic loops, whose decoupling is known to expose the central SOD1 ß-barrel triggering aggregation. Structural mapping of the preferential apo SOD1 Ca(2+) binding locations reveals that among the most frequent ligands for Ca(2+) are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. Calcium interactions thus diminish gatekeeping roles of these residues, by shielding repulsive interactions via stacking between aggregating ß-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in ALS inclusions. Interestingly, many fALS mutations occur at these positions, disclosing how Ca(2+) interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic ALS pathomechanisms.

Steric confinement and enhanced local flexibility assist knotting in simple models of protein folding.

The chaperonin complex GroEL-GroES is able to accelerate the folding process of knotted proteins considerably. However, the folding mechanism inside the chaperonin cage is elusive. Here we use a combination of lattice and off-lattice Monte Carlo simulations of simple Go models to study the effect of physical confinement and local flexibility on the folding process of protein model systems embedding a trefoil knot in their native structure. This study predicts that steric confinement plays a specific role in the folding of knotted proteins by increasing the knotting probability for very high degrees of confinement. This effect is observed for protein MJ0366 even above the melting temperature for confinement sizes compatible with the size of the GroEL/GroES chaperonin cage. An enhanced local flexibility produces the same qualitative effects on the folding process. In particular, we observe that knotting probability increases up to 40% in the transition state of protein MJ0366 when flexibility is enhanced. This is underlined by a structural change in the transition state, which becomes devoid of helical content. No relation between the knotting mechanism and flexibility was found in the context of the off-lattice model adopted in this work.

A simulated intermediate state for folding and aggregation provides insights into ΔN6 ß2-microglobulin amyloidogenic behavior.

A major component of ex vivo amyloid plaques of patients with dialysis-related amyloidosis (DRA) is a cleaved variant of ß2-microglobulin (ΔN6) lacking the first six N-terminal residues. Here we perform a computational study on ΔN6, which provides clues to understand the amyloidogenicity of the full-length ß2-microglobulin. Contrary to the wild-type form, ΔN6 is able to efficiently nucleate fibrillogenesis in vitro at physiological pH. This behavior is enhanced by a mild acidification of the medium such as that occurring in the synovial fluid of DRA patients. Results reported in this work, based on molecular simulations, indicate that deletion of the N-terminal hexapeptide triggers the formation of an intermediate state for folding and aggregation with an unstructured strand A and a native-like core. Strand A plays a pivotal role in aggregation by acting as a sticky hook in dimer assembly. This study further predicts that the detachment of strand A from the core is maximized at pH 6.2 resulting into higher aggregation efficiency. The structural mapping of the dimerization interface suggests that Tyr10, His13, Phe30 and His84 are hot-spot residues in ΔN6 amyloidogenesis.

How determinant is N-terminal to C-terminal coupling for protein folding?

This work investigates the role of N- to C- termini coupling in the folding transition of small, single domain proteins via extensive Monte Carlo simulations of both lattice and off-lattice models. The reported results provide compelling evidence that the existence of native interactions between the terminal regions of the polypeptide chain (i.e. termini coupling) is a major determinant of the height of the free energy barrier that separates the folded from the denatured state in a two-state folding transition, therefore being a critical modulator of protein folding rates and thermodynamic cooperativity. We further report that termini interactions are able to substantially modify the kinetic behavior dictated by the full set of native interactions. Indeed, a native structure of high contact order with "switched-off" termini-interactions actually folds faster than its circular permutant of lowest CO.

Effects of knot type in the folding of topologically complex lattice proteins.

The folding properties of a protein whose native structure contains a 52 knot are investigated by means of extensive Monte Carlo simulations of a simple lattice model and compared with those of a 31 knot. A 52 knot embedded in the native structure enhances the kinetic stability of the carrier lattice protein in a way that is clearly more pronounced than in the case of the 31 knot. However, this happens at the expense of a severe loss in folding efficiency, an observation that is consistent with the relative abundance of 31 and 52 knots in the Protein Data Bank. The folding mechanism of the 52 knot shares with that of the 31 knot the occurrence of a threading movement of the chain terminus that lays closer to the knotted core. However, co-concomitant knotting and folding in the 52 knot occurs with negligible probability, in sharp contrast to what is observed for the 31 knot. The study of several single point mutations highlights the importance in the folding of knotted proteins of the so-called structural mutations (i.e., energetic perturbations of native interactions between residues that are critical for knotting but not for folding). On the other hand, the present study predicts that mutations that perturb the folding transition state may significantly enhance the kinetic stability of knotted proteins provided they involve residues located within the knotted core.

Interplay between native topology and non-native interactions in the folding of tethered proteins.

We assessed the interplay of native topology and non-native interactions on surface-tethered protein folding via extensive Monte Carlo simulations of a simple lattice model. In particular, we investigated the thermodynamics and kinetics of protein-like sequences enclosing different amounts of non-native interactions to protein energetics, and which were designed to fold to distinct native topologies. Our results show that the high-contact order (CO) structure renders a folding transition that is robust to (external) steric constraints and non-native interactions. On the other hand, the folding process of the simple low-CO topology can be easily hampered by the presence of a nearby chemically inert plane. In this case, if non-native interactions are highly conspicuous during folding they can actually drive chain collapse into a very native-like trapped state, which impedes the formation of the native structure. The analysis of folding kinetics reveals that the empirical correlation between folding rate and CO may not apply to surface-tethered protein folding. Indeed, results reported here show that depending on the native environment of the tethered chain terminus the folding rate of a low-CO topology can become so drastically small that the high-CO topology actually folds faster under the same conditions. We predict that complex topologies are more likely to conserve their bulk folding mechanism upon surface tethering.

Structural and energetic determinants of co-translational folding.

We performed extensive lattice Monte Carlo simulations of ribosome-bound stalled nascent chains (RNCs) to explore the relative roles of native topology and non-native interactions in co-translational folding of small proteins. We found that the formation of a substantial part of the native structure generally occurs towards the end of protein synthesis. However, multi-domain structures, which are rich in local interactions, are able to develop gradually during chain elongation, while those with proximate chain termini require full protein synthesis to fold. A detailed assessment of the conformational ensembles populated by RNCs with different lengths reveals that the directionality of protein synthesis has a fine-tuning effect on the probability to populate low-energy conformations. In particular, if the participation of non-native interactions in folding energetics is mild, the formation of native-like conformations is majorly determined by the properties of the contact map around the tethering terminus. Likewise, a pair of RNCs differing by only 1-2 residues can populate structurally well-resolved low energy conformations with significantly different probabilities. An interesting structural feature of these low-energy conformations is that, irrespective of native structure, their non-native interactions are always long-ranged and marginally stabilizing. A comparison between the conformational spectra of RNCs and chain fragments folding freely in the bulk reveals drastic changes amongst the two set-ups depending on the native structure. Furthermore, they also show that the ribosome may enhance (up to 20%) the population of low energy conformations for chains folding to native structures dominated by local interactions. In contrast, a RNC folding to a non-local topology is forced to remain largely unstructured but can attain low energy conformations in bulk.

Assessing the effect of loop mutations in the folding space of ß2-microglobulin with molecular dynamics simulations.

We use molecular dynamics simulations of a full atomistic Go model to explore the impact of selected DE-loop mutations (D59P and W60C) on the folding space of protein human ß2-microglobulin (Hß2m), the causing agent of dialysis-related amyloidosis, a conformational disorder characterized by the deposition of insoluble amyloid fibrils in the osteoarticular system. Our simulations replicate the effect of mutations on the thermal stability that is observed in experiments in vitro. Furthermore, they predict the population of a partially folded state, with 60% of native internal free energy, which is akin to a molten globule. In the intermediate state, the solvent accessible surface area increases up to 40 times relative to the native state in 38% of the hydrophobic core residues, indicating that the identified species has aggregation potential. The intermediate state preserves the disulfide bond established between residue Cys25 and residue Cys80, which helps maintain the integrity of the core region, and is characterized by having two unstructured termini. The movements of the termini dominate the essential modes of the intermediate state, and exhibit the largest displacements in the D59P mutant, which is the most aggregation prone variant. PROPKA predictions of pKa suggest that the population of the intermediate state may be enhanced at acidic pH explaining the larger amyloidogenic potential observed in vitro at low pH for the WT protein and mutant forms.

A Specific Set of Heterogeneous Native Interactions Yields Efficient Knotting in Protein Folding.

Native interactions are crucial for folding, and non-native interactions appear to be critical for efficiently knotting proteins. Therefore, it is important to understand both their roles in the folding of knotted proteins. It has been proposed that non-native interactions drive the correct order of contact formation, which is essential to avoid backtracking and efficiently self-tie. In this study, we ask if non-native interactions are strictly necessary to tangle a protein or if the correct order of contact formation can be assured by a specific set of native, but otherwise heterogeneous (i.e., having distinct energies), interactions. In order to address this problem, we conducted extensive Monte Carlo simulations of lattice models of protein-like sequences designed to fold into a preselected knotted conformation embedding a trefoil knot. We were able to identify a specific set of heterogeneous native interactions that drives efficient knotting and is able to fold the protein when combined with the remaining native interactions modeled as homogeneous. This specific set of heterogeneous native interactions is strictly enough to efficiently self-tie. A distinctive feature of these native interactions is that they do not backtrack because their energies ensure the correct order of contact formation. Furthermore, they stabilize a knotted intermediate state, which is en route to the native structure. Our results thus show that-at least in the context of the adopted model-non-native interactions are not necessary to knot a protein. However, when they are taken into account in protein energetics, it is possible to find specific, nonlocal non-native interactions that operate as a scaffold that assists the knotting step.

Predicting stable binding modes from simulated dimers of the D76N mutant of ß 2-microglobulin.

The D76N mutant of the ß 2 m protein is a biologically motivated model system to study protein aggregation. There is strong experimental evidence, supported by molecular simulations, that D76N populates a highly dynamic conformation (which we originally named I 2 ) that exposes aggregation-prone patches as a result of the detachment of the two terminal regions. Here, we use Molecular Dynamics simulations to study the stability of an ensemble of dimers of I 2 generated via protein-protein docking. MM-PBSA calculations indicate that within the ensemble of investigated dimers the major contribution to interface stabilization at physiological pH comes from hydrophobic interactions between apolar residues. Our structural analysis also reveals that the interfacial region associated with the most stable binding modes are particularly rich in residues pertaining to both the N- and C-terminus, as well residues from the BC- and DE-loops. On the other hand, the less stable interfaces are stabilized by intermolecular interactions involving residues from the CD- and EF-loops. By focusing on the most stable binding modes, we used a simple geometric rule to propagate the corresponding dimer interfaces. We found that, in the absence of any kind of structural rearrangement occurring at an early stage of the oligomerization pathway, some interfaces drive a self-limited growth process, while others can be propagated indefinitely allowing the formation of long, polymerized chains. In particular, the interfacial region of the most stable binding mode reported here falls in the class of self-limited growth.

The folding of knotted proteins: insights from lattice simulations.

We carry out systematic Monte Carlo simulations of Go lattice proteins to investigate and compare the folding processes of two model proteins whose native structures differ from each other due to the presence of a trefoil knot located near the terminus of one of the protein chains. We show that the folding time of the knotted fold is larger than that of the unknotted protein and that this difference in folding time is particularly striking in the temperature region below the optimal folding temperature. Both proteins display similar folding transition temperatures, which is indicative of similar thermal stabilities. By using the folding probability reaction coordinate as an estimator of folding progression we have found out that the formation of the knot is mainly a late folding event in our shallow knot system.

The protein folding transition state: insights from kinetics and thermodynamics.

We perform extensive lattice Monte Carlo simulations of protein folding to construct and compare the equilibrium and the kinetic transition state ensembles of a model protein that folds to the native state with two-state kinetics. The kinetic definition of the transition state is based on the folding probability analysis method, and therefore on the selection of conformations with 0.4

The Early Phase of ß2-Microglobulin Aggregation: Perspectives From Molecular Simulations.

Protein ß2-microglobulin is the causing agent of two amyloidosis, dialysis related amyloidosis (DRA), affecting the bones and cartilages of individuals with chronic renal failure undergoing long-term hemodialysis, and a systemic amyloidosis, found in one French family, which impairs visceral organs. The protein's small size and its biomedical significance attracted the attention of theoretical scientists, and there are now several studies addressing its aggregation mechanism in the context of molecular simulations. Here, we review the early phase of ß2-microglobulin aggregation, by focusing on the identification and structural characterization of monomers with the ability to trigger aggregation, and initial small oligomers (dimers, tetramers, hexamers etc.) formed in the so-called nucleation phase. We focus our analysis on results from molecular simulations and integrate our views with those coming from in vitro experiments to provide a broader perspective of this interesting field of research. We also outline directions for future computer simulation studies.

Cooperativity and the origins of rapid, single-exponential kinetics in protein folding.

The folding of naturally occurring, single-domain proteins is usually well described as a simple, single-exponential process lacking significant trapped states. Here we further explore the hypothesis that the smooth energy landscape this implies, and the rapid kinetics it engenders, arises due to the extraordinary thermodynamic cooperativity of protein folding. Studying Miyazawa-Jernigan lattice polymers, we find that, even under conditions where the folding energy landscape is relatively optimized (designed sequences folding at their temperature of maximum folding rate), the folding of protein-like heteropolymers is accelerated when their thermodynamic cooperativity is enhanced by enhancing the nonadditivity of their energy potentials. At lower temperatures, where kinetic traps presumably play a more significant role in defining folding rates, we observe still greater cooperativity-induced acceleration. Consistent with these observations, we find that the folding kinetics of our computational models more closely approximates single-exponential behavior as their cooperativity approaches optimal levels. These observations suggest that the rapid folding of naturally occurring proteins is, in part, a consequence of their remarkably cooperative folding.