Unraveling the Role of Hydrogen Bonds in Thrombin via Two Machine Learning Methods

Hydrogen bonds play a critical role in the folding and stability of proteins, such as proteins and nucleic acids, by providing strong and directional interactions. They help to maintain the secondary and 3D structure of proteins, and structural changes in these molecules often result from the formation or breaking of hydrogen bonds. To gain insights into these hydrogen bonding networks, we applied two machine learning models - a logistic regression model and a decision tree model - to study four variants of thrombin: wild-type, ΔK9, E8K, and R4A. Our results showed that both models have their unique advantages. The logistic regression model highlighted potential key residues (GLU295) in thrombin’s allosteric pathways, while the decision tree model identified important hydrogen bonding motifs. This information can aid in understanding the mechanisms of folding in proteins and has potential applications in drug design and other therapies. The use of these two models highlights their usefulness in studying hydrogen bonding networks in proteins

All-Atom Molecular Dynamics Reveals Mechanism of Zinc Complexation with Therapeutic F10

Advancing the use of therapeutic nucleic acids requires understanding the chemical and structural properties that allow these polymers to promote the death of malignant cells. Here we explore Zn²⁺ complexation by the fluoropyrimidine polymer F10, which has strong activities in multiple preclinical models of cancer. Delivery of fluoropyrimidine FdUMP in the 10-residue polymer F10 rather than the nucleobase (5-fluorouracil) allows consideration of metal ion binding effects on drug delivery. The differences in metal ion interactions with fluoropyrimidine compared to normal DNA results in conformation changes that affect protein binding, cell uptake, and codelivery of metals such as zinc, and the cytoxicity thereof. Microsecond-time-scale, all-atom simulations of F10 predict that zinc selectively stabilizes the polymer via interactions with backbone phosphate groups and suggest a mechanism of complexation for the zinc-base interactions shown in previous experimental work. The positive zinc ions are attracted to the negatively charged phosphate groups. Once the Zn²⁺ ions are near F10, they cause the base’s N3 nitrogen to deprotonate. Subsequently, magnesium atoms displace zinc from their interactions with phosphate, freeing the zinc ions to interact with the FdU bases by forming weak interactions with the O4 oxygen and the fluorine attached to C5. These interactions of magnesium with phosphate groups and zinc with nucleobases agree with previous experimental results and are seen in MD simulations only when magnesium is introduced after N3 deprotonation, indicating a specific order of metal binding events. Additionally, we predict interactions between zinc and F10’s O2 atoms, which were not previously observed. By comparison to 10mers of polyU and polydT, we also predict that the presence of fluorine increases the binding affinity of zinc to F10 relative to analogous strands of RNA and DNA consisting of only native nucleotides

All-Atom MD Predicts Magnesium-Induced Hairpin in Chemically Perturbed RNA Analog of F10 Therapeutic

Given their increasingly frequent usage, understanding the chemical and structural properties which allow therapeutic nucleic acids to promote the death of cancer cells is critical for medical advancement. One molecule of interest is a 10-mer of FdUMP (5-fluoro-2′-deoxyuridine-5′-O-monophosphate) also called F10. To investigate causes of structural stability, we have computationally restored the 2′ oxygen on each ribose sugar of the phosphodiester backbone, creating FUMP[10]. Microsecond time-scale, all-atom, simulations of FUMP[10] in the presence of 150 mM MgCl₂ predict that the strand has a 45% probability of folding into a stable hairpin-like secondary structure. Analysis of 16 μs of data reveals phosphate interactions as likely contributors to the stability of this folded state. Comparison with polydT and polyU simulations predicts that FUMP[10]’s lowest order structures last for one to 2 orders of magnitude longer than similar nucleic acid strands. Here we provide a brief structural and conformational analysis of the predicted structures of FUMP[10], and suggest insights into its stability via comparison to F10, polydT, and polyU

Binding Site Configurations Probe the Structure and Dynamics of the Zinc Finger of NEMO (NF-κB Essential Modulator)

Zinc-finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. Here, we investigate how binding site protonation states and zinc coordination influence protein structure, dynamics, and ultimately function, as these pivotal regulatory proteins are increasingly important for protein engineering and therapeutic discovery. To better understand the thermodynamics and dynamics of the zinc finger of NEMO (NF-κB essential modulator), as well as the role of zinc, we present results of 20 μs molecular dynamics trajectories, 5 μs for each of four active site configurations. Consistent with experimental evidence, the zinc ion is essential for mechanical stabilization of the functional, folded conformation. Hydrogen bond motifs are unique for deprotonated configurations yet overlap in protonated cases. Correlated motions and principal component analysis corroborate the similarity of the protonated configurations and highlight unique relationships of the zinc-bound configuration. We hypothesize a potential mechanism for zinc binding from results of the thiol configurations. The deprotonated, zinc-bound configuration alone predominantly maintains its tertiary structure throughout all 5 μs and alludes rare conformations potentially important for (im)­proper zinc-finger-related protein–protein or protein–DNA interactions

Uncovering Large-Scale Conformational Change in Molecular Dynamics without Prior Knowledge

As the length of molecular dynamics (MD) trajectories grows with increasing computational power, so does the importance of clustering methods for partitioning trajectories into conformational bins. Of the methods available, the vast majority require users to either have some <i>a priori</i> knowledge about the system to be clustered or to tune clustering parameters through trial and error. Here we present non-parametric uses of two modern clustering techniques suitable for first-pass investigation of an MD trajectory. Being non-parametric, these methods require neither prior knowledge nor parameter tuning. The first method, HDBSCAN, is fastrelative to other popular clustering methodsand is able to group unstructured or intrinsically disordered systems (such as intrinsically disordered proteins, or IDPs) into bins that represent global conformational shifts. HDBSCAN is also useful for determining the overall stability of a systemas it tends to group stable systems into one or two binsand identifying transition events between metastable states. The second method, iMWK-Means, with explicit rescaling followed by K-Means, while slower than HDBSCAN, performs well with stable, structured systems such as folded proteins and is able to identify higher resolution details such as changes in relative position of secondary structural elements. Used in conjunction, these clustering methods allow a user to discern quickly and without prior knowledge the stability of a simulated system and identify both local and global conformational changes

Site-Specific DNA–Doxorubicin Conjugates Display Enhanced Cytotoxicity to Breast Cancer Cells

Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-¹⁵N]-2′-deoxyguanosine in conjunction with [¹H–¹⁵N] 2D NMR, while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life >30 h, considerably longer than alternative covalent and noncovalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anticancer drugs