To assess the sampling quality, we used autocorrelation functions of a typical MD simulation. A good sampling is indicated by a convergence of the autocorrelation function with an MD time that is proportional to the correlation time.
We analyzed the conformational dynamics of K6- and K11-diUbs using the CGK63 model (two Ub monomers linked by a K48-G76 isopeptide bond) and CGM1 model (two Ub monomers linked by an M1-G76 peptide bond, also denoted as linear diUbs). Our simulations indicate that both of these diUbs are highly dependent on pH like K48-diUb, and decreasing pH will increase their populations in open conformations.
Molecular Structure
Our simulation results reveal that both the proximal and distal Ub interfaces consist of basic residues which can form electrostatically repulsive force as they are buried at the interface. Distal Ub interface comprises T7-G10, Q40 and R42 whereas proximal Ub interface is composed of K6-G10, E34-P37, I44 and G47. These features are consistent with experimental observations that the proximal sidechain is closer to the apex than the distal.
We found that the free energy landscape of K63-diUb and M1-diUb is similar to each other but distinct from that of K48-diUb. This suggests that topological constraint imposed by different linkage types does not have a significant effect on the functional landscape.
The free energy surfaces of the compact structures of diUbs with different linkage types were projected as function of centroid distance () and RMSD from the available experimental structures solved by NMR and X-ray crystallography. The grey regions indicate the conformational region occupied by the experimental structures.
From the viewpoint of energy landscape, the formation of a covalent bond between two Ub monomers makes them form compact structures. This is consistent with the binding landscape view which suggests that forming a covalent bond shifts the population of pre-existing states in a protein. Our simulation results also show that hydrophobic interactions made a much larger contribution to the formation of compact diUbs than electrostatic interactions, especially for K11-, K6- and K48-linked diUbs.
Biological Function
The small gene family of oligopeptide transporters (OPT) act as proton-coupled symporters that transport small peptides and amino acids including glutathione. These membrane proteins possess 12 transmembrane domains and several signature motifs that distinguish them from other protein families such as metal-chelate transporters. OPTs are involved in diverse biological functions such as long distance metal mobilization, nitrogen mobilization, heavy metal sequestration, and soil scavenging (Bogs et al., 2003). For more details please visit cgk 33
Ubiquitin (Ub) is a multifunctional modification protein that marks proteins for degradation or for other regulatory functions including signaling, DNA repair and autophagy. The majority of Ub-dependent protein degradation is mediated by ubiquitin E3 ligases, which are responsible for the recognition and binding of target proteins to ubiquitin molecules (Ubi) in a covalent reaction (Kehl et al., 2015).
The ubiquitin linkage residues are close to each other in both sequence and configuration space. Therefore, a large part of the functional landscape is forbidden on the binding landscape for diUbs containing K27-, K29- and K33-linked lysines due to specific topological constraints. However, the atomic details of these functional landscapes are poorly understood. In this study, we use a combination of computational and experimental techniques to reveal the key molecular mechanisms that govern these topological constraint-imposed functional landscapes.