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Reddem, E.

Publications and source records attributed to Reddem, E..

4 recordsLinked to original sources
bioRxiv2026

A functional investigation of antibody Fc-FcRn variant binding guided by *in silico* free energy perturbation methods

Accurate calculation of energy changes upon mutation is a key requirement for the effective use of computational methods in protein design. In this study, we applied free energy perturbation (FEP) calculations to predict the effects of mutations on the binding free energy between the immunoglobulin subtype G (IgG) antibody fragment-crystallizable (Fc) region and the neonatal Fc receptor (FcRn), an interaction that is primarily responsible for antibody half-life. We assembled an extensive experimental dataset of Fc-FcRn binding affinities for wild-type (wt) and mutant complexes, including values from literature and from newly measured results. Starting from a crystal structure of the M252Y/S254T/T256E ("YTE") Fc variant bound to FcRn, we prepared all-atom models of human IgG1-subtype wt and YTE variant Fc-FcRn complexes, adding explicit hydrogens and assigning protonation states for key ionizable residues. Initial results using standard FEP protocols to compute relative binding free energies were promising but exhibited multiple outliers. By accounting for coupling effects for FEP mutations near key histidine residues, we improved the results for several outliers, suggesting such coupling as an important approach for pH-sensitive systems. Further, upon determining new crystal structures of four Fc variants at multiple pH values, we observed subtle conformational changes in unbound Fc; by accounting for these conformational changes in FEP calculations, we additionally improved agreement with experiment. The detailed structural and energetic analyses of the Fc-FcRn system we present here thus provide an accurate energy-calculation framework to enable rational in silico design of novel Fc variants. SignificanceThe ability to determine changes in binding affinity upon mutation is critical to structure-based protein design. In this study, we demonstrate a successful computational approach using free energy perturbation (FEP) calculations on the antibody Fc-FcRn complex, a medically relevant system with implications for both therapeutic and prophylactic antibody use. Our successful calculation of accurate binding energies across a wide range of cases speaks to the power of the FEP methodology in navigating the free energy landscapes of dynamic molecular complexes. Furthermore, we show that accurate Fc-FcRn affinity calculations required careful consideration of conformational flexibility between bound and unbound states, contributing to our functional understanding of a system that will be important for future rational antibody-design efforts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/721095v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f754d8org.highwire.dtl.DTLVardef@1e366beorg.highwire.dtl.DTLVardef@6e67caorg.highwire.dtl.DTLVardef@602a14_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗
bioRxiv2021

Isolation and comparative analysis of antibodies that broadly neutralize sarbecoviruses

The devastation caused by SARS-CoV-2 has made clear the importance of pandemic preparedness. To address future zoonotic outbreaks due to related viruses in the sarbecovirus subgenus, we identified a human monoclonal antibody, 10-40, that neutralized or bound all sarbecoviruses tested in vitro and protected against SARS-CoV-2 and SARS-CoV in vivo. Comparative studies with other receptor-binding domain (RBD)-directed antibodies showed 10-40 to have the greatest breadth against sarbecoviruses and thus its promise as an agent for pandemic preparedness. Moreover, structural analyses on 10-40 and similar antibodies not only defined an epitope cluster in the inner face of the RBD that is well conserved among sarbecoviruses, but also uncovered a new antibody class with a common CDRH3 motif. Our analyses also suggested that elicitation of this class of antibodies may not be overly difficult, an observation that bodes well for the development of a pan-sarbecovirus vaccine. One sentence summaryA monoclonal antibody that neutralizes or binds all sarbecoviruses tested and represents a reproducible antibody class.

microbiology↗
bioRxiv2021

Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest or termination, involving the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The dsRNAs are then released from the Pol IV-RDR2 complex and diced into siRNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 [A], determined by cryo-electron microscopy. The N-terminal region contains an RNA-recognition motif (RRM) adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1-2 nucleotides (nt) internal to the 3 ends of its templates and can transcribe the RNA of an RNA-DNA hybrid provided that 9 or more nucleotides at the RNAs 3 end is unpaired. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3 end occurs as the DNA template and non-template strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and transcribe the second strand. SignificanceRDR2 is critical for siRNA-directed DNA methylation in Arabidopsis, functioning in physical association with DNA-dependent Pol IV to synthesize the second strands of double-stranded siRNA precursors. Basepairing between the DNA template strand transcribed by Pol IV and the nontemplate DNA strand is known to induce Pol IV arrest and Pol IV-RDR2 transcriptional coupling, but how this occurs is unknown. We report the structure of RDR2 and experimental evidence for how RDR2 engages its RNA templates and initiates transcription. RDR2 engages the ends of RNAs displaced from RNA-DNA hybrids, suggesting a model in which Pol IV arrest and backtracking, accompanied by DNA strand reannealing, extrudes the 3 end of the Pol IV transcript, allowing RNA engagement and second-strand synthesis.

molecular biology↗
bioRxiv2019

A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity

Toxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB can perform both ribosome-dependent and -independent RNA cleavage. We have also characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence of the ribosome. The toxin concentration inside bacterial cells is unknown and according to current models, should increase upon activation of YoeB through degradation of the YefM antitoxin. The discovery of general nuclease activity by AtYoeB, and perhaps other YoeB toxins, offers an opportunity to explore the plasticity of this protein fold and its potential role in the evolution of nucleases.

biochemistry↗