Zirconium Coordination Chemistry and Its Role in Optimizing Hydroxymate Chelation: Insights from Molecular Dynamics.

In the past decade, there has been a growth in using Zirconium-89 (89Zr) as a radionuclide in nuclear medicine for cancer diagnostic imaging and drug discovery processes. Although one of the most popular chelators for 89Zr, desferrioxamine (DFO) is typically presented as a hexadentate ligand, our work suggests a different scenario. The coordination structure of the Zr4+-DFO complex has primarily been informed by DFT-based calculations, which typically ignore temperature and therefore entropic and dynamic solvent effects. In this work, free energy calculations using molecular dynamics simulations, where the conformational fluctuations of both the ligand and the solvent are explicitly included, are used to compare the binding of Zr4+ cations with two different chelators, DFO and 4HMS, the latter of which is an octadentate ligand that has been recently proposed as a better chelator due to the presence of four hydroxymate groups. We find that thermally induced disorder leads to an open hexadentate chelate structure of the Zr4+-DFO complex, leaving the Zr4+ metal exposed to the solvent. A stable coordination of Zr4+ with 4HMS, however, is formed by involving both hydroxamate groups and water molecules in a more closely packed structure.

A Rosetta-based protein design protocol converging to natural sequences.

Computational protein design has emerged as a powerful tool capable of identifying sequences compatible with pre-defined protein structures. The sequence design protocols, implemented in the Rosetta suite, have become widely used in the protein engineering community. To understand the strengths and limitations of the Rosetta design framework, we tested several design protocols on two distinct folds (SH3-1 and Ubiquitin). The sequence optimization, when started from native structures and natural sequences or polyvaline sequences, converges to sequences that are not recognized as belonging to the fold family of the target protein by standard bioinformatic tools, such as BLAST and Hmmer. The sequences generated from both starting conditions (native and polyvaline) are instead very similar to each other and recognized by Hmmer as belonging to the same "family." This demonstrates the capability of Rosetta to converge to similar sequences, even when sampling from distinct starting conditions, but, on the other hand, shows intrinsic inaccuracy of the scoring function that drifts toward sequences that lack identifiable natural sequence signatures. To address this problem, we developed a protocol embedding Rosetta Design simulations in a genetic algorithm, in which the sequence search is biased to converge to sequences that exist in nature. This protocol allows us to obtain sequences that have recognizable natural sequence signatures and, experimentally, the designed proteins are biochemically well behaved and thermodynamically stable.

Candidate Binding Sites for Allosteric Inhibition of the SARS-CoV-2 Main Protease from the Analysis of Large-Scale Molecular Dynamics Simulations.

We analyzed a 100 µs MD trajectory of the SARS-CoV-2 main protease by a non-parametric data analysis approach which allows characterizing a free energy landscape as a simultaneous function of hundreds of variables. We identified several conformations that, when visited by the dynamics, are stable for several hundred nanoseconds. We explicitly characterize and describe these metastable states. In some of these configurations, the catalytic dyad is less accessible. Stabilizing them by a suitable binder could lead to an inhibition of the enzymatic activity. In our analysis we keep track of relevant contacts between residues which are selectively broken or formed in the states. Some of these contacts are formed by residues which are far from the catalytic dyad and are accessible to the solvent. Based on this analysis we propose some relevant contact patterns and three possible binding sites which could be targeted to achieve allosteric inhibition.

Explicit Characterization of the Free-Energy Landscape of a Protein in the Space of All Its Cα Carbons.

By using an approach that allows computing the free energy in high-dimensional spaces together with a clustering technique capable of identifying kinetic attractors stabilized by conformational disorder, we analyze a molecular dynamics trajectory of the Villin headpiece from Lindorff-Larsen, K.; et al. How fast-folding proteins fold. Science 2011, 334, 517-520. We compute its free-energy landscape in the space of all its Cα carbons. This landscape has the shape of a 12-dimensional funnel with the free energy decreasing monotonically as a function of the native contacts. There are no significant folding barriers. The funnel can be partitioned in five regions, three mainly folded and two unfolded, which behave as Markov states. The slowest relaxation time among these states corresponds to the folding transition. The second slowest time is only twice smaller and corresponds to a transition within the unfolded state. This indicates that the unfolded part of the funnel has a nontrivial shape, which induces a sizable kinetic barrier between disordered states.

Stabilization of epigenetic states of CpG islands by local cooperation.

DNA methylation of CpG sites is an important epigenetic mark in mammals. Active promoters are often associated with unmethylated CpG sites, whereas methylated CpG sites correlate with silenced promoters. Methylation of CpG sites must be generally described as a dynamical process that is mediated by methylation enzymes, such as DNMT1 and DNMT3a/b. However, there are several models of how CpG sites can be protected from methylation and thereby remain unmethylated. In this paper we examine the combination of both: the positive feedbacks of DNA methylation and a short range counterpart which in turn protects-and thereby maintains-the unmethylated state. The emergent dynamics is provided by collaborative, re-enforcing feedbacks in favor of methylated CpG islands and cooperative protection of one CpG site by another in favor of unmethylated CpG sites. Our results suggest that this synthesis of mechanisms provides equally robust maintenance of both the unmethylated and methylated states of CpG islands.