Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases.

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestral ribonuclease (AncRNase) from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

Mechanism of nucleotide discrimination by the translesion synthesis polymerase Rev1.

Rev1 is a translesion DNA synthesis (TLS) polymerase involved in the bypass of adducted-guanine bases and abasic sites during DNA replication. During damage bypass, Rev1 utilizes a protein-template mechanism of DNA synthesis, where the templating DNA base is evicted from the Rev1 active site and replaced by an arginine side chain that preferentially binds incoming dCTP. Here, we utilize X-ray crystallography and molecular dynamics simulations to obtain structural insight into the dCTP specificity of Rev1. We show the Rev1 R324 protein-template forms sub-optimal hydrogen bonds with incoming dTTP, dGTP, and dATP that prevents Rev1 from adopting a catalytically competent conformation. Additionally, we show the Rev1 R324 protein-template forms optimal hydrogen bonds with incoming rCTP. However, the incoming rCTP adopts an altered sugar pucker, which prevents the formation of a catalytically competent Rev1 active site. This work provides novel insight into the mechanisms for nucleotide discrimination by the TLS polymerase Rev1.

Structure-mechanics statistical learning uncovers mechanical relay in proteins.

A protein's adaptive response to its substrates is one of the key questions driving molecular physics and physical chemistry. This work employs the recently developed structure-mechanics statistical learning method to establish a mechanical perspective. Specifically, by mapping all-atom molecular dynamics simulations onto the spring parameters of a backbone-side-chain elastic network model, the chemical moiety specific force constants (or mechanical rigidity) are used to assemble the rigidity graph, which is the matrix of inter-residue coupling strength. Using the S1A protease and the PDZ3 signaling domain as examples, chains of spatially contiguous residues are found to exhibit prominent changes in their mechanical rigidity upon substrate binding or dissociation. Such a mechanical-relay picture thus provides a mechanistic underpinning for conformational changes, long-range communication, and inter-domain allostery in both proteins, where the responsive mechanical hotspots are mostly residues having important biological functions or significant mutation sensitivity.

Altered APE1 activity on abasic ribonucleotides is mediated by changes in the nucleoside sugar pucker.

Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard nucleotide in the human genome. Typically, misinserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage. However, rNTPs are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Additionally, rAPs have been found in nascent RNA and coupled to R-loops. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is responsible for the initial processing of rAPs embedded in DNA and in R-loops. APE1 is a well characterized AP endonuclease that cleaves 5' of abasic sites, but its ability to cleave at rAPs remains poorly understood. Here, we utilize enzyme kinetics, X-ray crystallography, and molecular dynamics simulations to provide insight into rAP processing by APE1. Enzyme kinetics were used to determine pre-steady-state rates of APE1 cleavage on DNA substrates containing rAP, revealing a decrease in activity compared to cleavage at a canonical deoxy-AP substrate. Using X-ray crystallography, we identified novel contacts between the rAP and the APE1 active site. We demonstrate that the rAP sugar pucker is accommodated in the active site in a C3'-endo conformation, influencing its position and contributing to a decrease in activity compared to the deoxy-AP site. Together, this work provides molecular level insights into rAP processing by APE1 and advances our understanding of ribonucleotide processing within genomic DNA.

Importance of electrostatic polarizability in calculating cysteine acidity constants and copper(I) binding energy of Bacillus subtilis CopZ.

CopZ is a copper chaperone from Bacillus subtilis. It is an important part of Cu(I) trafficking. We have calculated pK(a) values for the CXXC motif of this protein, which is responsible for the Cu(I) binding, and the Cu(I) binding constants. Polarizable and fixed-charges formalisms were used, and solvation parameters for the both models have been refitted. We had to partially redevelop parameters for the protonated and deprotonated cysteine residues. We have discovered that the polarizable force field (PFF) is qualitatively superior and allows a uniformly better level of energetic results. The PFF pK(a) values for cysteine are within about 0.8-2.8 pH units of the experimental data, while the fixed-charges OPLS formalism yields errors of up to tens of units. The PFF magnitude of the copper binding energy is about 10 kcal/mol or 50% higher than the experimental value, while the using the refitted OPLS parameters leads to an overall positive binding energy, thus predicting no thermodynamically stable complex. At the same time, the agreement of the polarizable S···Cu(I) distances with the experimental results is within 0.08 Å range, and the nonpolarizable calculations lead to an error of about 0.4 Å. Moreover, the accuracy of the PFF has been achieved without any explicit fitting to either pK(a) or CopZ···Cu(I) binding energies. We believe that this makes our polarizable technique a choice method in reproducing protein-copper binding and further supports the notion that explicit treatment of electrostatic polarization is crucial in many biologically relevant studies, especially ion binding and transport.

Electrostatic polarization is crucial in reproducing Cu(I) interaction energies and hydration.

We have explored the suitability of fixed-charges and polarizable force fields for modeling interactions of the monovalent Cu(I) ion. Parameters for this ion have been tested and refitted within the fixed-charges OPLS-AA and polarizable force field (PFF) frameworks. While this ion plays an important role in many protein interactions, the attention to it in developing empirical force fields is limited. Our PFF parameters for the copper ion worked very well for the Cu(I) interactions with water, while both the original OPLS2005 and our refitted OPLS versions moderately underestimated the copper-water interaction energy. However, the greatest problem in using the nonpolarizable fixed-charges OPLS force field was observed while calculating interaction energies and distances for Cu(I)-benzene complexes. The OPLS2005 model underestimates the interaction energy by a factor of 4. Refitting the OPLS parameters reduced this underestimation to a factor of 2.2-2.4, but only at a cost of distorting the complex geometry. At the same time, the polarizable calculations had an error of about 4%. Moreover, we then used the PFF and nonpolarizable refitted OPLS models for finding free energy of hydration for copper ion via molecular dynamics simulations. While the OPLS calculations lead to a 22% error in the solvation energy, the PFF result was off by only 1.8%. This was achieved with no refitting of the parameters but simply by employing the model developed for the Cu(I) interaction with a single water molecule. We believe that the presented results not only lead to a conclusion about a qualitatively greater suitability of polarizable force fields for simulating molecular interactions with ions but also attest to the excellent level of transferability of PFF parameters.

Quality of random number generators significantly affects results of Monte Carlo simulations for organic and biological systems.

We have simulated pure liquid butane, methanol, and hydrated alanine polypeptide with the Monte Carlo technique using three kinds of random number generators (RNG's)-the standard Linear Congruential Generator (LCG), a modification of the LCG with additional randomization used in the BOSS software, and the "Mersenne Twister" generator by Matsumoto and Nishimura. While using the latter two RNG's leads to reasonably similar physical features, the LCG produces significant different results. For the pure fluids, a noticeable expansion occurs. Using the original LCG on butane yields, a molecular volume of 171.4 Å(3) per molecule compared to about 163.6-163.9 Å(3) for the other two generators, a deviation of about 5%. For methanol, the LCG produces an average volume of 86.3 Å(3) per molecule, which is about 24% higher than the 68.8-70.2 Å(3) obtained with the RNG's in BOSS and the generator by Matsumoto and Nishimura. In case of the hydrated tridecaalanine peptide, the volume and energy tend to be noticeably greater with the LCG than with the BOSS (modified LCG) RNG's. For the simulated hydrated extended conformation of tridecaalanine, the difference in volume reached about 87%. The uniformity and periodicity of the generators do not seem to play the crucial role in these phenomena. We conclude that, it is important to test a RNG's by modeling a system such as the pure liquid methanol with a well-established force field before routinely employing it in Monte Carlo simulations.

Intrinsically disordered proteins in a physics-based world.

Intrinsically disordered proteins (IDPs) are a newly recognized class of functional proteins that rely on a lack of stable structure for function. They are highly prevalent in biology, play fundamental roles, and are extensively involved in human diseases. For signaling and regulation, IDPs often fold into stable structures upon binding to specific targets. The mechanisms of these coupled binding and folding processes are of significant importance because they underlie the organization of regulatory networks that dictate various aspects of cellular decision-making. This review first discusses the challenge in detailed experimental characterization of these heterogeneous and dynamics proteins and the unique and exciting opportunity for physics-based modeling to make crucial contributions, and then summarizes key lessons from recent de novo simulations of the structure and interactions of several regulatory IDPs.

Reproducing basic pKa values for turkey ovomucoid third domain using a polarizable force field.

We have extended our previous studies of calculating acidity constants for the acidic residues found in the turkey ovomucoid third domain protein (OMTKY3) by determining the relative pKa values for the basic residues (Lys13, Arg21, Lys29, Lys34, His52, and Lys55). A polarizable force field (PFF) was employed. The values of the pKa were found by direct comparison of energies of solvated protonated and deprotonated forms of the protein. Poisson-Boltzmann (PBF) and surface generalized Born (SGB) continuum solvation models represent the hydration, and a nonpolarizable fixed-charge OPLS-AA force field was used for comparison. Our results indicate that (i) the pKa values of the basic residues can be found in close agreement with the experimental values when a PFF is used in conjunction with the PBF solvation model, (ii) it is sufficient to take into the account only the residues which are in close proximity (hydrogen bonded) to the residue in question, and (iii) the PBF solvation model is superior to the SGB solvation model for these pKa calculations. The average error with the PBF/PFF model is only 0.7 pH unit, compared with 2.2 and 6.1 units for the PBF/OPLS and SGB/OPLS, respectively. The maximum deviation of the PBF/PFF results from the experimental values is 1.7 pH units compared with 6.0 pH units for the PBF/OPLS. Moreover, the best results were obtained while using an advanced nonpolar energy calculation scheme. The overall conclusion is that this methodology and force field are suitable for the accurate assessment of pKa shifts for both acidic and basic protein residues.