Characterization of Monobody Scaffold Interactions with Ligand via Force Spectroscopy and Steered Molecular Dynamics

Inosine-5′-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities. Thereby, the comparison of their high-resolution structures defines the mechanistic and structural details of a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity of eukaryotic IMPDHs. Remarkably, retinopathy-associated mutations lie within the mechanical hinges of the conformational change, highlighting its physiological relevance. Our results expand the mechanistic repertoire of Bateman domains and pave the road to new approaches targeting IMPDHs.

Section: Methodsmentioning

confidence: 99%

A nucleotide-controlled conformational switch modulates the activity of eukaryotic IMP dehydrogenases

Buey

Fernandez-Justel

Marcos‐Alcalde

et al. 2017

“…MD trajectory analysis considered hydrogen bonds and salt bridges formation as well as RMSD of the biomolecules. Hydrogen bonds were quantified via Visual Molecular Dynamics (VMD) 45 Hbonds plugin following computational protocols applied for similar antibody systems in the literature 29,30,46 . A hydrogen bond was quantified when the distance between a hydrogen donor (D) and an acceptor atom (A) was shorter than 3.5 Å as well as the angle H-D-A was shorter than 60.0°.…”

Section: Methodsmentioning

confidence: 99%

Antibody-mediated biorecognition of myelin oligodendrocyte glycoprotein: computational evidence of demyelination-related epitopes

Ierich

Brum

Moraes

et al. 2019

Antigen-antibody interaction is crucial in autoimmune disease pathogenesis, as multiple sclerosis and neuromyelitis optica. Given that, autoantibodies are essential biomolecules, of which the myelin oligodendrocyte glycoprotein (MOG) can figure as a target. Here we combined Molecular Dynamics (MD), Steered Molecular Dynamics (SMD), and Atomic Force Microscope (AFM) to detail MOG recognition by its specific antibody. The complex model consisted of the MOG external domain interacting with an experimental anti-MOG antibody from the Protein Data Bank (1PKQ). Computational data demonstrated thirteen MOG residues with a robust contribution to the antigen-antibody interaction. Comprising five of the thirteen anchor residues (ASP102, HIS103, SER104, TYR105, and GLN106), the well-known MOG92–106 peptide in complex with the anti-MOG was analysed by AFM and SMD. These analyses evidenced similar force values of 780 pN and 765 pN for computational and experimental MOG92–106 and anti-MOG detachment, respectively. MOG92–106 was responsible for 75% of the total force measured between MOG external domain and anti-MOG, holding the interaction with the antibody. The antigen-antibody binding was confirmed by Surface Plasmon Resonance (SPR) measurements. Combined approaches presented here can conveniently be adjusted to detail novel molecules in diseases research. This can optimize pre-clinical steps, guiding experiments, reducing costs, and animal model usage.

“…To ensure good thermalization of the system after the modification of the water box, a heating protocol similar to that used in the free MD simulations was applied. During each SMD trajectory, the centres of mass of Smc1A-head and Smc3-head were forced to separate 32.5 Å at a constant velocity over 13 ns (2.5 Å ns −1 ) with a spring constant of 5 kcal mol −1 Å −2 , in the range of conditions used in similar SMD studies 72 , 73 . The separation distance was kept constant for 0.1 ns at the start and end of each trajectory to better describe the quasi-equilibrium states at both ends.…”

Section: Methodsmentioning

confidence: 99%

Two-step ATP-driven opening of cohesin head

Marcos‐Alcalde

Mendieta‐Moreno

Puisac³

et al. 2017

The cohesin ring is a protein complex composed of four core subunits: Smc1A, Smc3, Rad21 and Stag1/2. It is involved in chromosome segregation, DNA repair, chromatin organization and transcription regulation. Opening of the ring occurs at the “head” structure, formed of the ATPase domains of Smc1A and Smc3 and Rad21. We investigate the mechanisms of the cohesin ring opening using techniques of free molecular dynamics (MD), steered MD and quantum mechanics/molecular mechanics MD (QM/MM MD). The study allows the thorough analysis of the opening events at the atomic scale: i) ATP hydrolysis at the Smc1A site, evaluating the role of the carboxy-terminal domain of Rad21 in the process; ii) the activation of the Smc3 site potentially mediated by the movement of specific amino acids; and iii) opening of the head domains after the two ATP hydrolysis events. Our study suggests that the cohesin ring opening is triggered by a sequential activation of the ATP sites in which ATP hydrolysis at the Smc1A site induces ATPase activity at the Smc3 site. Our analysis also provides an explanation for the effect of pathogenic variants related to cohesinopathies and cancer.