Paul S. Nerenberg

Homogeneous and heterogeneous tertiary structure ensembles of amyloid-β peptides.

The interplay of modern molecular simulation and high-quality nuclear magnetic resonance (NMR) experiments has reached a fruitful stage for quantitative characterization of structural ensembles of disordered peptides. Amyloid-β 1-42 (Aβ42), the primary peptide associated with Alzheimers disease, and fragments such as Aβ21-30 are both classified as intrinsically disordered peptides (IDPs). We use a variety of NMR observables to validate de novo molecular dynamics simulations in explicit water to characterize the tertiary structure ensemble of Aβ42 and Aβ21-30 from the perspective of their classification as IDPs. Unlike the Aβ21-30 fragment that conforms to expectations of an IDP that is primarily extended, we find that Aβ42 samples conformations reflecting all possible secondary structure categories and spans the range of IDP classifications from collapsed structured states to highly extended conformations, making it an IDP with a far more heterogeneous tertiary ensemble.

Optimizing Solute–Water van der Waals Interactions To Reproduce Solvation Free Energies

An accurate representation of solute-water interactions is necessary for molecular dynamics simulations of biomolecules that reside in aqueous environments. Modern force fields and advanced water models describe solute-solute and water-water interactions reasonably accurately but have known shortcomings in describing solute-water interactions, demonstrated by the large differences between calculated and experimental solvation free energies across a range of peptide and drug chemistries. In this work, we introduce a method for optimizing solute-water van der Waals interactions to reproduce experimental solvation free energy data and apply it to the optimization of a fixed charge force field (AMBER ff99SB/GAFF) and advanced water model (TIP4P-Ew). We show that, with these optimizations, the combination of AMBER ff99SB/GAFF and TIP4P-Ew is able to reproduce the solvation free energies of a variety of biologically relevant small molecules to within 1.0 k(B)T. We further validate these optimizations by examining the aggregation propensities of dipeptide-water solutions, the conformational preferences of short disordered peptides, and the native state stability and dynamics of a folded protein.

Perturbing the folding energy landscape of the bacterial immunity protein Im7 by site-specific N-linked glycosylation

N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the bacterial immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol-1. In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition.

Cleavage site specificity and conformational selection in type I collagen degradation.

Excessive degradation of type I collagen is associated with a variety of human diseases such as arthritis, tumor metastasis, and atherosclerosis. Methods that further our understanding of collagenolysis may therefore provide insights into the mechanism of several important disorders. Prior experiments suggest that cleavage of collagen in vitro requires intact full-length collagenase, a multidomain protein containing both a catalytic and a hemopexin-like domain. In this work we demonstrate that type I collagen can be degraded at room temperature, a temperature well below the melting temperature of type I collagen, by collagenase deletion mutants that only contain the catalytic domain of the enzyme. Furthermore, these mutant enzymes hydrolyze the same peptide bond that is recognized by the corresponding full-length enzymes. Hence enzyme specificity at room temperature is achieved without the hemopexin-like domain. We demonstrate that these findings can be explained in light of a conformational selection mechanism that dictates that collagenases preferentially recognize and cleave preformed partially unfolded states of collagen.

Do collagenases unwind triple‐helical collagen before peptide bond hydrolysis? Reinterpreting experimental observations with mathematical models

It has been postulated that triple‐helical collagen is actively unwound by collagenases before peptide bond hydrolysis—a supposition that explains the small catalytic rate constant associated with collagenolysis. We propose an alternate model of collagen degradation that does not require active unwinding by collagenases, but instead suggests that the regions of collagen near the collagenase cleavage site can adopt either a native triple‐helical or a partially unfolded conformation. In this model, collagenases preferentially bind to and stabilize partially unfolded conformers before cleaving the scissile bond. Existing experimental observations (which were previously taken to support active unwinding models) are reinterpreted using corroborative evidence from numerical simulations and found to be consistent with this framework. These data support the notion that collagen, like all other biological heteropolymers, undergoes thermal fluctuations that cause it to sample distinct structures in the neighborhood of the native state, and collagenolysis occurs when collagenases recognize the appropriate unwound conformers. Proteins 2008.

Differential Unfolding of α1 and α2 Chains in Type I Collagen and Collagenolysis

Collagenolysis plays a central role in many disease processes and a detailed understanding of the mechanism of collagen degradation is of immense interest. While a considerable body of information about collagenolysis exists, the details of the underlying molecular mechanism are unclear. Therefore, to further our understanding of the precise mechanism of collagen degradation, we used molecular dynamics simulations to explore the structure of human type I collagen in the vicinity of the collagenase cleavage site. Since post-translational proline hydroxylation is an important step in the synthesis of collagen chains, we used the DNA sequence for the alpha1 and alpha2 chains of human type I collagen, and the known amino acid sequences for bovine and chicken type I collagen, to infer which prolines are hydroxylated in the vicinity of the collagenase cleavage site. Simulations of type I collagen in this region suggest that partial unfolding of the alpha2 chain is energetically preferred relative to unfolding of alpha1 chains. Localized unfolding of the alpha2 chain leads to the formation of a structure that has disrupted hydrogen bonds N-terminal to the collagenase cleavage site. Our data suggest that this disruption in hydrogen bonding pattern leads to increased chain flexibility, thereby enabling the alpha2 chain to sample different partially unfolded states. Surprisingly, our data also imply that alpha2 chain unfolding is mediated by the non-hydroxylation of a proline residue that is N-terminal to the cleavage site in alpha1 chains. These results suggest that hydroxylation on one chain (alpha1) can affect the structure of another chain (alpha2), and point to a critical role for the non-hydroxylation of proline residues near the collagenase cleavage site.

Optimizing Protein–Protein van der Waals Interactions for the AMBER ff9x/ff12 Force Field

The quality of molecular dynamics (MD) simulations relies heavily on the accuracy of the underlying force field. In recent years, considerable effort has been put into developing more accurate dihedral angle potentials for MD force fields, but relatively little work has focused on the nonbonded parameters, many of which are two decades old. In this work, we assess the accuracy of protein-protein van der Waals interactions in the AMBER ff9x/ff12 force field. Across a test set of 44 neat organic liquids containing the moieties present in proteins, we find root-mean-square (RMS) errors of 1.26 kcal/mol in enthalpy of vaporization and 0.36 g/cm(3) in liquid densities. We then optimize the van der Waals radii and well depths for all of the relevant atom types using these observables, which lowers the RMS errors in enthalpy of vaporization and liquid density of our validation set to 0.59 kcal/mol (53% reduction) and 0.019 g/cm(3) (46% reduction), respectively. Limitations in our parameter optimization were evident for certain atom types, however, and we discuss the implications of these observations for future force field development.

Disordered Structural Ensembles of Vasopressin and Oxytocin and Their Mutants

Vasopressin and oxytocin are intrinsically disordered cyclic nonapeptides belonging to a family of neurohypophysial hormones. Although unique in their functions, these peptides differ only by two residues and both feature a tocin ring formed by the disulfide bridge between first and sixth cysteine residues. This sequence and structural similarity are experimentally linked to oxytocin agonism at vasopressin receptors and vasopressin antagonism at oxytocin receptors. Yet single- or double-residue mutations in both peptides have been shown to have drastic impacts on their activities at either receptor, and possibly the ability to bind to their neurophysin carrier protein. In this study we perform molecular dynamics simulations of the unbound native and mutant sequences of the oxytocin and vasopressin hormones to characterize their structural ensembles. We classify the subpopulations of these structural ensembles on the basis of the distributions of radius of gyration and secondary structure and hydrogen-bonding features of the canonical tocin ring and disordered tail region. We then relate the structural changes observed in the unbound form of the different hormone sequences to experimental information about peptide receptor binding, and more indirectly, carrier protein binding affinity, receptor activity, and protease degradation. This study supports the hypothesis that the structural characteristics of the unbound form of an IDP can be used to predict structural or functional preferences of its functional bound form.

Physical basis of metal-binding specificity in Escherichia coli NikR.

In Escherichia coli and other bacteria, nickel uptake is regulated by the transcription factor NikR. Nickel binding at high-affinity sites in E. coli NikR (EcNikR) facilitates EcNikR binding to the nik operon, where it then suppresses transcription of genes encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal-binding site is also present when NikR binds to the nik operon. Moreover, this co-crystal structure raises the question of what metal occupies the second site under physiological conditions: K(+), which is present in the crystal structure, or Ni(2+), which has been proposed to bind to low- as well as high-affinity sites on EcNikR. To determine which ion is preferred at the second metal-binding site and the physical basis for any preference of one ion over another in both the second metal-binding site and the high-affinity sites, we conducted a series of detailed molecular simulations on the EcNikR structure. Simulations that place Ni(2+) at high-affinity sites lead to stable trajectories with realistic ion-ligand distances and geometries, while simulations that place K(+) at these sites lead to conformational changes in the protein that are likely unfavorable for ion binding. By contrast, simulations on the second metal site in the EcNikR-DNA complex lead to stable trajectories with realistic geometries regardless of whether K(+) or Ni(2+) occupies this site. Electrostatic binding free energy calculations, however, suggest that EcNikR binding to DNA is more favorable when the second metal-binding site contains K(+). An analysis of the energetic contributions to the electrostatic binding free energy suggests that, while the interaction between EcNikR and DNA is more favorable when the second site contains Ni(2+), the large desolvation penalty associated with moving Ni(2+) from solution to the relatively buried second site offsets this favorable interaction term. Additional free energy simulations that account for both electrostatic and non-electrostatic effects argue that EcNikR binding to DNA is most favorable when the second site contains a monovalent ion the size of K(+). Taken together, these data suggest that the EcNikR structure is most stable when Ni(2+) occupies high-affinity sites and that EcNikR binding to DNA is more favorable when the second site contains K(+).

New developments in force fields for biomolecular simulations

Biomolecular force field development has been instrumental in improving the predictive power of molecular simulations over the past four decades. More recently, the era of large quantitative experimental datasets and ubiquitous high performance computing power has enabled rapid progress in the field. In this review we summarize recent developments in all-atom protein, nucleic acid, and small molecule force fields, paying specific attention to developments in parameterization methods and improvements in the representations of nonbonded interactions that are critical for solving the challenging biophysical problems of the present. We also sketch out new avenues for force field development and grand challenge applications for the near future.