Biology subjects

MacKerell, A.

Publications and source records attributed to MacKerell, A..

4 recordsLinked to original sources

bioRxiv2021

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain site are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains, as well as the overall flexibility of EndoS2, to the proteins catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulation and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and long-time MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models. Author SummaryThe pathogen Streptococcus pyogenes uses the endoglycosidases S and S2 to cleave the glycans on the Fc portion of IgG antibodies, leading to a decreased cytotoxicity of the antibodies, thereby evading the host immune response. To identify potential structures of the complex of EndoS2 with IgG that could lead to the catalytic hydrolysis of the IgG glycan, molecular modeling and molecular dynamics simulations were applied. The resulting structural models predict that EndoS2 initially interacts through its carbohydrate binding module (CBM) with the IgG with subsequent interactions with the catalytic glycoside hydrolase (GH) domain yielding stable complexes. In the modeled complexes the CBM and the GH interact either simultaneously with the same Fc CH2/CH3 domain or with the two individual Fc CH2/CH3 domains separately to yield potentially catalytically competent species. In addition, apo EndoS2 is shown to assume both open and closed conformations allowing it to directly form either type of complex from which deglycosylation of either mono- or diglycosylated IgG species may occur. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/445403v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@15ffa95org.highwire.dtl.DTLVardef@3c8032org.highwire.dtl.DTLVardef@452bdorg.highwire.dtl.DTLVardef@1d3f85a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Contributions and competition of Mg2+ and K+ in folding and stabilization of the Twister Ribozyme

Native folded and compact intermediate states of RNA typically involve tertiary structures in the presence of divalent ions such as Mg2+ in a background of monovalent ions. In a recent study we showed how the presence of Mg2+ impacts the transition from partially unfolded to folded states through a "push-pull" mechanism where the ion both favors and disfavors the sampling of specific phosphate-phosphate interactions. To better understand the ion atmosphere of RNA in folded and partially folded states results from atomistic Umbrella Sampling and oscillating chemical potential Grand Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) simulations are used to obtain atomic-level details of the distributions of Mg2+ and K+ ions around Twister RNA. Results show the presence of 100 mM Mg2+ to lead to increased charge neutralization over that predicted by counterion condensation theory. Upon going from partially unfolded to folded states overall charge neutralization increases at all studied ion concentrations that, while associated with an increase in the number of direct ion-phosphate interactions, is fully accounted for by the monovalent K+ ions. Furthermore, K+ preferentially interacts with purine N7 atoms of helical regions in partially unfolded states thereby potentially stabilizing them. Thus, both secondary helical structures and formation of tertiary structures leads to increased counterion condensation, thereby stabilizing those structural features of Twister. Notably, it is shown that K+ can act as a surrogate for Mg2+ by participating in specific interactions with non-sequential phosphate pairs that occur in the folded state, explaining the ability of Twister to self-cleave at sub-millimolar Mg2+ concentrations.

biophysics↗

bioRxiv2019

Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Non-Sequential Phosphate Pairs

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg2+, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg2+ in folded conformations of RNA have been studied extensively, however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) simulations are used to capture the energetics and atomic-level details of Mg2+-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg2+, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg2+ with two or more non-sequential phosphate groups. Notably, the present results indicate a push-pull mechanism where the Mg2+-RNA interactions actually lead to destabilization of specific non-sequential phosphate-phosphate interactions while other interactions are stabilized, a balance that facilitates the folding and stabilization of Twister including formation hydrogen bonds associated with the tertiary structure. The present study establishes a better understanding of how Mg2+ ion-interactions contribute to RNA structural properties and stability.\n\nStatement of SignificanceRNAs are biologically and therapeutically of great emerging interest such that it is critical to understand how RNA molecules fold into complex structures. While experiments yield information on the stabilization of RNA by ions they are limited in the atomic-level insights they can provide. A combination of enhanced sampling methods is applied to explore the compact intermediate states of RNA and their interactions with Mg2+ ions. Results reveal a picture of how Mg2+ overall stabilizes short phosphate-phosphate interactions thereby facilitating the stabilization of RNA, though doing so by both the stabilization and destabilization of specific interactions. The applied method will be applicable to exploring the impact of divalent ions on the conformational heterogeneity of a range of macromolecules.

biophysics↗

bioRxiv2018

Impact of branching on the conformational heterogeneity of the lipopolysaccharide from Klebsiella pneumoniae: Implications for vaccine design

Resistance of Klebsiella pneumoniae (KP) to antibiotics has motivated the development of an efficacious KP human vaccine that would not be subject to antibiotic resistance. Klebsiella lipopolysaccharide (LPS) associated O polysaccharide (OPS) types have provoked broad interest as a vaccine antigen as there are only 4 that predominate worldwide (O1, O2a, O3, O5). Klebsiella O1 and O2 OPS are polygalactans that share a common D-Gal-I structure, for which a variant D-Gal-III was recently discovered. To understand the potential impact of this variability on antigenicity, a detailed molecular picture of the conformational differences associated with the addition of the D-Gal-III (1[->]4)Gal branch is presented using enhanced-sampling molecular dynamics simulations. In D-Gal-I two major conformational states are observed while the presence of the 1[->]4 branch in D-Gal-III resulted in only a single dominant extended state. Stabilization of the more folded states in D-Gal-I is due to a O4-H...O2 hydrogen bond in the linear backbone that cannot occur in D-Gal-III as the O4 is in the Galp(1[->]4)Galp glycosidic linkage. The impact of branching in D-Gal-III also significantly decreases the accessibility of the monosaccharides in the linear backbone region of D-Gal-I, while the accessibility of the terminal D-Gal-II region of the OPS is not substantially altered. The present results suggest that a vaccine that targets both the D-Gal-I and D-Gal-III LPS can be developed by using D-Gal-III as the antigen combined with cross-reactivity experiments using the Gal-II polysaccharide to assure that this region of the LPS is the primary epitope of the antigen.\n\nAuthor SummaryKlebsiella pneumoniae (KP) is a bacterial pathogen commonly associated with hospital acquired infections to antibiotics and is of increasing concern due to the development of resistance to antibiotics including those of last resort. Development of an efficacious KP human vaccine would not be subject to the mechanisms governing antibiotic resistance and, is thus, a public health priority. The present study applies computer simulations to understand the effects of branching on the antigenic D-Gal-I and D-Gal-III O polysaccharide (OPS) species of KP. Results show that branching leads to a single dominate extended conformation, though that conformation occurs in both species. In addition, the terminal D-Gal-II region of both OPS sample similar, exposed conformations while regions of the central repeating units of the OPS are potential hindered from interactions with antibodies in the presence of the branched D-Gal-III OPS. The results suggest a strategy for vaccine development based on D-Gal-III as the antigen combined with cross reactivity experiments to assure that the D-Gal-II region is the primary epitope of the antigen.

biophysics↗