ff99SB Force Field Research Articles

Accurate PDZ/Peptide Binding Specificity with Additive and Polarizable Free Energy Simulations

PDZ domains contain 80–100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.

Validation of molecular force field parameters for peptides including isomerized amino acids.

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age-related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l-α-amino acids, the accuracy of the computational results for d-amino acids and β-amino acids was comparable to those for l-α-amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.

Role of the Chalcogen (S, Se, Te) in the Oxidation Mechanism of the Glutathione Peroxidase Active Site.

The oxidation by H2 O2 of the human phospholipid hydroperoxide glutathione peroxidase (GPx4), used as a model peroxidase selenoenzyme, as well as that of its cysteine (Cys) and tellurocysteine (Tec) mutants, was investigated in silico through a combined classic and quantum mechanics approach to assess the role of the different chalcogens. To perform this analysis, new parameters for selenocysteine (Sec) and tellurocysteine (Tec) were accurately derived for the AMBER ff14SB force field. The oxidation represents the initial step of the antioxidant activity of GPx, which catalyzes the reduction of H2 O2 and organic hydroperoxides by glutathione (GSH). A mechanism involving a charge-separation intermediate is feasible for the Cys and Sec enzymes, leading from the initial thiol/selenol form to sulfenic/selenenic acid, whereas for the Tec mutant a direct oxidation pathway is proposed. Activation strain analyses, performed for Cys-GPx and Sec-GPx, provided insight into the rate-accelerating effect of selenium as compared to sulfur and the role of specific amino acids other than Cys/Sec that are typically conserved in the catalytic pocket.

Simple MD-based model for oxidative folding of peptides and proteins

Significant strides have been recently made to fold peptides and small proteins in silico using MD simulations. However, facilities are currently lacking to include disulfide bonding in the MD models of protein folding. To address this problem, we have developed a simple empirical protocol to model formation of disulfides, which is perturbation-free, retains the same speed as conventional MD simulations and allows one to control the reaction rate. The new protocol has been tested on 15-aminoacid peptide guanylin containing four cysteine residues; the net simulation time using Amber ff14SB force field was 61 μs. The resulting isomer distribution is in qualitative agreement with experiment, suggesting that oxidative folding of guanylin in vitro occurs under kinetic control. The highly stable conformation of the so-called isomer 2(B) has been obtained for full-length guanylin, which is significantly different from the poorly ordered structure of the truncated peptide PDB ID 1GNB. In addition, we have simulated oxidative folding of guanylin within the 94-aminoacid prohormone proguanylin. The obtained structure is in good agreement with the NMR coordinates 1O8R. The proposed modeling strategy can help to explore certain fundamental aspects of protein folding and is potentially relevant for manufacturing of synthetic peptides and recombinant proteins.

In silico investigation on the inhibition of Aβ42 aggregation by Aβ40 peptide by potential of mean force study

Recent experimental data revealed that small, soluble Amyloid beta (Aβ42) oligomers, especially dimers impair synaptic plasticity and memory leading to Alzheimer’s disease. Here, we have studied dimerization of Aβ42/Aβ42 homo-dimer and Aβ40/Aβ42 hetero-dimer in terms of free energy profile by all-atom simulations using the ff99SB force field. We have found that in the presence of Aβ40 peptide, there exists a strong tendency to form a hetero-dimer with Aβ42 peptide, suggesting that a possible co-oligomerization. Furthermore, we have investigated the effects of Aβ40 on the Aβ42 peptide. Our study also shows that in presence of Aβ40, the beta-content of Aβ42 monomer is reduced. Additionally, certain residues important for bending in Aβ42 peptide attained an increased flexibility in the presence of Aβ40. The salt-bridge destabilization also manifested the impact of Aβ40 on Aβ42 peptide as a whole. Based on this, one may expect that Aβ40 inhibits the aggregation propensity of Aβ42. Moreover, the binding free energy obtained by the molecular mechanics–Poisson–Boltzmann surface area method also revealed a strong affinity between the two isoforms thereby suggests that Aβ40 binding induces conformational change in Aβ42. Our results suggest that co-oligomerization of Aβ isoforms may play a substantial role in Alzheimer’s disease.

Structure-Guided Protein Transition Modeling with a Probabilistic Roadmap Algorithm.

Proteins are macromolecules in perpetual motion, switching between structural states to modulate their function. A detailed characterization of the precise yet complex relationship between protein structure, dynamics, and function requires elucidating transitions between functionally-relevant states. Doing so challenges both wet and dry laboratories, as protein dynamics involves disparate temporal scales. In this paper, we present a novel, sampling-based algorithm to compute transition paths. The algorithm exploits two main ideas. First, it leverages known structures to initialize its search and define a reduced conformation space for rapid sampling. This is key to address the insufficient sampling issue suffered by sampling-based algorithms. Second, the algorithm embeds samples in a nearest-neighbor graph where transition paths can be efficiently computed via queries. The algorithm adapts the probabilistic roadmap framework that is popular in robot motion planning. In addition to efficiently computing lowest-cost paths between any given structures, the algorithm allows investigating hypotheses regarding the order of experimentally-known structures in a transition event. This novel contribution is likely to open up new venues of research. Detailed analysis is presented on multiple-basin proteins of relevance to human disease. Multiscaling and the AMBER ff14SB force field are used to obtain energetically-credible paths at atomistic detail.

MD Modeling of Oxidative Folding in Peptides and Proteins

We are now at the dawn of the new era in protein modeling when it becomes possible to fold proteins in silico “from the first principles”, i.e. using unbiased MD simulations. We have developed an empirical protocol to include the process of disulfide bonding into conventional (unrestrained) MD models of protein folding. The protocol has been tested on 15-amino-acid peptide guanylin, containing four cysteine residues. The fifty guanylin trajectories recorded with GPU-enabled Amber 14 program under ff14SB force field produced thirty eight disulfide-bonded folds, with the isomer distribution that is broadly similar to the one observed experimentally. The PBSA/GBSA energy calculations performed on these MD models suggest that the isomer distribution is under kinetic rather than thermodynamic control. We have also used the new protocol to simulate disulfide bonding in the terminal guanylin segment within the 94-amino-acid prohormone proguanylin. It is envisaged that in future an efficient reactive force field can be constructed along these lines to model protein oxidative folding.

Ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB.

Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but also improved secondary structure content in small peptides and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.

Residue-Specific Force Field (RSFF2) Improves the Modeling of Conformational Behavior of Peptides and Proteins.

A recent report of (3)J(HNHα) scalar coupling constants for hundreds of two-residue peptides has provided an important opportunity to test simulation force fields for proteins. Here, we compare the abilities of three derivatives of the Amber ff99SB force field to reproduce these data. We report molecular dynamics (MD) simulations of 256 two-residue peptides and show that the recently developed residue-specific force field (RSFF2) produces a dramatic improvement in the agreement with experimental (3)J(HNHα) coupling constants. We further show that RSFF2 also appears to produce a modest improvement in reproducing the (3)J(HNHα) coupling constants of five model proteins. Perhaps surprisingly, an analysis of neighboring residue effects (NREs) on the (3)J(HNHα) coupling constants of the two-residue peptides indicates little difference between the force fields' abilities to reproduce experimental NREs. We speculate that this might indicate limitations in the force fields' descriptions of nonbonded interactions between adjacent side chains or with terminal capping groups.

Restoring histone deacetylase activity by waste product release. A view from molecular mechanics simulations with mammalian HDAC8.

HDAC8 is a Zn(II) -based, single-peptide mammalian histone deacetylase that is localized mainly in the cytoskeleton of smooth muscle cells, thus regulating muscle contractility. HDACs are also widely involved in cellular processes, ranging from cell differentiation to proliferation, senescence, and apoptosis; in particular, protecting a telomerase activator from ubiquitin-mediated degradation. How HDACs can eliminate the hydrolytic reaction products, in order that the process of deacetylation of the acetyllysine moiety of histones can take place again, has long been debated in the scientific literature, without reaching any firm conclusion, however. This question is the subject of the present work, carried out along a theoretical line that is capable of describing the whole pathway followed by the acetate product (ACT). A model was built here on the crystal data for the Y306F-mutated HDAC8 complex with a diacetylated peptide of the p53-tumor-suppressor class. That was followed by manually hydrolyzing the acetylated moiety bound to Zn(II) and discharging the monoacetylated peptide product (MAP). The latter was replaced by a H2 O molecule bound to Zn(II) , while ACT was left free in the reaction cage. This Zn(II) cluster was DFT-parameterized for the ff99SB force field without any further bias. As the result of random-acceleration molecular dynamics (RAMD) simulations, egress of ACT from the reaction cage toward the aqueous environment can follow three pathways. Two of them utilize the channel for peptide (or histone) uptake and are preferred, if ACT leaves the reaction center before MAP (or the deacetylated histone). The third pathway, developing along the internal channel, is available to ACT even if MAP is still in place.

QM/MM simulations of amyloid-β 42 degradation by IDE in the presence and absence of ATP.

The ability of the insulin-degrading enzyme (IDE) to degrade amyloid-β 42 (Aβ42), a process regulated by ATP, has been studied as an alternative path in the development of drugs against Alzheimer's disease. In this study, we calculated the potential of mean force for the degradation of Aβ42 by IDE in the presence and absence of ATP by umbrella sampling with hybrid quantum mechanics and molecular mechanics (QM/MM) calculations, using the SCC-DFTB QM Hamiltonian and Amber ff99SB force field. Results indicate that the reaction occurs in two steps: The first step is characterized by the formation of the intermediate. The second step is characterized by breaking the peptide bond of the substrate, the latter being the rate-determining step. In our simulations, the activation energy barrier in the absence of ATP is 15 ± 2 kcal mol(-1), which is 7 kcal mol(-1) lower than in the presence of ATP, indicating that the presence of the nucleotide decreases the reaction rate by about 10(5) times.

Residue-specific force field based on protein coil library. RSFF2: modification of AMBER ff99SB.

Recently, we developed a residue-specific force field (RSFF1) based on conformational free-energy distributions of the 20 amino acid residues from a protein coil library. Most parameters in RSFF1 were adopted from the OPLS-AA/L force field, but some van der Waals and torsional parameters that effectively affect local conformational preferences were introduced specifically for individual residues to fit the coil library distributions. Here a similar strategy has been applied to modify the Amber ff99SB force field, and a new force field named RSFF2 is developed. It can successfully fold α-helical structures such as polyalanine peptides, Trp-cage miniprotein, and villin headpiece subdomain and β-sheet structures such as Trpzip-2, GB1 β-hairpins, and the WW domain, simultaneously. The properties of various popular force fields in balancing between α-helix and β-sheet are analyzed based on their descriptions of local conformational features of various residues, and the analysis reveals the importance of accurate local free-energy distributions. Unlike the RSFF1, which overestimates the stability of both α-helix and β-sheet, RSFF2 gives melting curves of α-helical peptides and Trp-cage in good agreement with experimental data. Fitting to the two-state model, RSFF2 gives folding enthalpies and entropies in reasonably good agreement with available experimental results.

Convergence and reproducibility in molecular dynamics simulations of the DNA duplex d(GCACGAACGAACGAACGC)

BackgroundThe structure and dynamics of DNA are critically related to its function. Molecular dynamics simulations augment experiment by providing detailed information about the atomic motions. However, to date the simulations have not been long enough for convergence of the dynamics and structural properties of DNA. MethodsMolecular dynamics simulations performed with AMBER using the ff99SB force field with the parmbsc0 modifications, including ensembles of independent simulations, were compared to long timescale molecular dynamics performed with the specialized Anton MD engine on the B-DNA structure d(GCACGAACGAACGAACGC). To assess convergence, the decay of the average RMSD values over longer and longer time intervals was evaluated in addition to assessing convergence of the dynamics via the Kullback–Leibler divergence of principal component projection histograms. ResultsThese molecular dynamics simulations—including one of the longest simulations of DNA published to date at ~44μs—surprisingly suggest that the structure and dynamics of the DNA helix, neglecting the terminal base pairs, are essentially fully converged on the ~1–5μs timescale. ConclusionsWe can now reproducibly converge the structure and dynamics of B-DNA helices, omitting the terminal base pairs, on the μs time scale with both the AMBER and CHARMM C36 nucleic acid force fields. Results from independent ensembles of simulations starting from different initial conditions, when aggregated, match the results from long timescale simulations on the specialized Anton MD engine. General significanceWith access to large-scale GPU resources or the specialized MD engine “Anton” it is possible for a variety of molecular systems to reproducibly and reliably converge the conformational ensemble of sampled structures. This article is part of a Special Issue entitled: Recent developments of molecular dynamics.

Computational design of a full-length model of HIV-1 integrase: modeling of new inhibitors and comparison of their calculated binding energies with those previously studied

A full-length model of integrase (IN) of the human immunodeficiency virus type 1 (HIV-1) was constructed based on the distinctly resolved X-ray crystal structures of its three domains, named N-terminal, catalytic core and C-terminal. Thirty-one already known inhibitors with varieties of structural differences as well as nine newly tested ones were docked into the catalytic core. The molecular dynamic (MD) and binding properties of these complexes were obtained by MD calculations. The binding energies calculated by molecular mechanic/Poisson Boltzmann solvation area were significantly correlationed with available IC50. Four inhibitors including two newly designed were also docked into the full-length model and their MD behaviors and binding properties were calculated. It was found that one of the newly designed compounds forms a better complex with HIV-1 IN compared to the rest including raltegravir. MD calculations were performed with AMBER suite of programs using ff99SB force field for the proteins and the general Amber force field for the ligands. In conclusion, the results have produced a promising standpoint not only in the construction of the full-length model but also in development of new drugs against it. However, the role of multimer formation and the involvement of DNAs, and their subsequent effect on the complexation and inhibition, are required to arrive at a conclusive decision.

Assessing the influence of solvation models on structural characteristics of intrinsically disordered protein

Intrinsically disordered proteins are associated with functions of signaling and regulation as well as with a number of diseases such as cancer and neurodegenerative conditions. Molecular dynamics simulation is potentially a powerful tool to study those protein conformational disorders in full atomic details that would be difficult to gain from experimental measurements. However, the accuracy of the simulation critically relies on the quality of underlying potential energy functions or force fields. In particular, structural properties of intrinsically disordered proteins are expected to depend more sensitively on protein–water interactions because of their increased solvent exposure than folded globular proteins. Here, we investigate the impact of solvation models on structural characteristics of full-length amyloid-beta (Aβ) protein, an intrinsically disordered protein whose aggregation is linked to Alzheimer’s disease. For this purpose, we performed extensive molecular dynamics simulations by combining AMBER ff99SB force field for protein with differing solvent water models as well as solvation procedures with different solvent box sizes. We find that the structural characteristics of Aβ protein differ remarkably depending on the water model adopted as well as on the solvent box size. Our results demonstrate the significant sensitivity on the solvation procedure including the solvent force field in characterizing structural properties of intrinsically disordered proteins.

Insights into the Folding and Unfolding Processes of Wild-Type and Mutated SH3 Domain by Molecular Dynamics and Replica Exchange Molecular Dynamics Simulations

Src-homology regions 3 (SH3) domain is essential for the down-regulation of tyrosine kinase activity. Mutation A39V/N53P/V55L of SH3 is found to be relative to the urgent misfolding diseases. To gain insight, the human and gallus SH3 domains (PDB ID: 1NYG and 2LP5), including 58 amino acids in each protein, were selected for MD simulations (Amber11, ff99SB force field) and cluster analysis to investigate the influence of mutations on the spatial structure of the SH3 domain. It is found that the large conformational change of mutations mainly exists in three areas in the vicinity of protein core: RT loop, N-src loop, distal β-hairpin to 310 helix. The C-terminus of the mutated gallus SH3 is disordered after simulation, which represents the intermediate state of aggregation. The disappeared strong Hbond net in the mutated human and gallus systems will make these mutated proteins looser than the wild-type proteins. Additionally, by performing the REMD simulations on the gallus SH3 domain, the mutated domain is found to have an obvious effect on the unfolding process. These studies will be helpful for further aggregation mechanisms investigations on SH3 family.

A Study of Intrinsically Disordered Proteins from Nuclear Pore Complex

Nuclear pore complex (NPC) is giant molecular assembly that acts as a highly selective gate. Its central channel is composed of FG repeat-containing nucleoporins (FG nups), which are natively disordered proteins that facilitate the passage of receptor-cargo complexes through the NPC. However, their mechanism to facilitate selective transport is still unknown. We carried out molecular dynamics simulations of single and multiple FG nups to unveil their dynamics and structures. As an important aspect of our studies of intrinsically disordered proteins (IDPs), we made extensive comparisons between two popular water models, TIP3P and TIP4P-Ew, in combination with Amber ff99sb force field to simulate different IDPs. We found that TIP4P-Ew combined with ff99sb gives better reproduction of experimental observations for FG nups. The reason is because of better solvation of water model to charged side chain of amino acid residues. However, we also identified a caveat of current force field by studying the mutated form of FG nups. We found that the shape of IDPs is much closer to a rod-like shape regardless of whether they are extended or collapsed, which could be a signature of IDPs. Then, we explored the interaction of multiple FG nups to show that the arrangement of the FG nups can be different depending on the inter-chain distances. We identified the influence from the types of FG nups and inter-molecular spacing. FG nups showed a strong dependence of inter-molecular interaction strength on molecular spacing. However, inter-molecular interaction for one type is weaker compared to that of another type. While most of the FG nups of the two types are mainly disordered despite inter- and intra- molecular interactions, we observed noticeable beta-sheet formation at certain inter-molecular spacings, indicating a significant role between inter-molecular interaction and secondary structure formation.

CONFORMATIONAL DYNAMICS OF HIV-1 PROTEASE: A COMPARATIVE MOLECULAR DYNAMICS SIMULATION STUDY WITH MULTIPLE AMBER FORCE FIELDS

Flap dynamics of HIV-1 protease (HIV-pr) controls the entry of inhibitors and substrates to the active site. Dynamical models from previous simulations are not all consistent with each other and not all are supported by the NMR results. In the present work, the effect of force field on the dynamics of HIV-pr is investigated by MD simulations using three AMBER force fields ff99, ff99SB, and ff03. The generalized order parameters for amide backbone are calculated from the three force fields and compared with the NMR S2 values. We found that the ff99SB and ff03 force field calculated order parameters agree reasonably well with the NMR S2 values, whereas ff99 calculated values deviate most from the NMR order parameters. Stereochemical geometry of protein models from each force field also agrees well with the remarks from NMR S2 values. However, between ff99SB and ff03, there are several differences, most notably in the loop regions. It is found that these loops are, in general, more flexible in the ff03 force field. This results in a larger active site cavity in the simulation with the ff03 force field. The effect of this difference in computer-aided drug design against flexible receptors is discussed.

Extension of the AMBER force field to cyclic α,α dialkylated peptides

The popular biomolecular AMBER (ff99SB) force field (FF) has been extended with new parameters for the simulations of peptides containing α,α dialkylated residues with cyclic side chains. Together with the recent set of nitroxide parameters [E. Stendardo, A. Pedone, P. Cimino, M. C. Menziani, O. Crescenzi and V. Barone, Phys. Chem. Chem. Phys., 2010, 12, 11697] this extension allows treating the TOAC residue (TOAC, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) widely used as a spin label in protein studies. All the conformational minima of the Ac-Ac(6)C-NMe (Ac = acetyl, Ac(6)C = 1-aminocyclohexaneacetic acid, NMe = methylamino) and Ac-TOAC-NMe dipeptides have been examined in terms of geometry and relative energy stability by Quantum Mechanical (QM) computations employing an hybrid density functional (PBE0) for an extended training set of conformers with various folds. A very good agreement between QM and MM (molecular mechanics) data has been obtained in most of the investigated properties, including solvent effects. Finally, the new set of parameters has been validated by comparing the conformational and dynamical behavior of TOAC-labeled polypeptides investigated by means of classical molecular dynamics (MD) simulations with QM data and experimental evidence. The new FF accurately describes the tuning of conformational and dynamical behavior of the Ac-TOAC-NMe dipeptide and double spin-labeled heptapeptide Fmoc-(Aib-Aib-TOAC)(2)-Aib-OMe (Fmoc, fluorenyl-9-methoxycarbonyl; Aib, α-aminoisobutyric acid; OMe, methoxy) by solvents with different polarity. In particular, we found that the 3(10) helical structure of heptapeptide is the most stable one in vacuo, with a geometry very similar to the X-ray crystallographic structure, whereas a conformational equilibrium between the 3(10)- and α-helical structures is established in aqueous solution, in agreement with EPR data.

Molecular Insights into DNA Polymerase Deterrents for Ribonucleotide Insertion

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase β discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn(2+) for Mg(2+) decreased sugar discrimination for both wild-type and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2' of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 Å, respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase β utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.