Engineering of conserved residues near antibody heavy chain complementary determining region 3 (HCDR3) improves both affinity and stability

Affinity and stability are crucial parameters in antibody development and engineering approaches. Although improvement in both metrics is desirable, trade-offs are almost unavoidable. Heavy chain complementarity determining region 3 (HCDR3) is the best-known region for antibody affinity but its impact on stability is often neglected. Here, we present a mutagenesis study of conserved residues near HCDR3 to elicit the role of this region in the affinity-stability trade-off. These key residues are positioned around the conserved salt bridge between VH-K94 and VH-D101 which is crucial for HCDR3 integrity. We show that the additional salt bridge at the stem of HCDR3 (VH-K94:VH-D101:VH-D102) has an extensive impact on this loop's conformation, therefore simultaneous improvement in both affinity and stability. We find that the disruption of π-π stacking near HCDR3 (VH-Y100E:VL-Y49) at the VH-VL interface cause an irrecoverable loss in stability even if it improves the affinity. Molecular simulations of putative rescue mutants exhibit complex and often non-additive effects. We confirm that our experimental measurements agree with the molecular dynamic simulations providing detailed insights for the spatial orientation of HCDR3. VH-V102 right next to HCDR3 salt bridge might be an ideal candidate to overcome affinity-stability trade-off

Unraveling linker histone interactions in nucleosomes

© 2021 Elsevier LtdConsiderable progress has been made recently in defining the interactions of linker histones (H1s) within nucleosomes. Major advancements include atomic resolution structures of the globular domain of full-length H1s in the context of nucleosomes containing full-length linker DNA. Although these studies have led to a detailed understanding of the interactions and dynamics of H1 globular domains in the canonical on-dyad nucleosome binding pocket, more information regarding the intrinsically disordered N-terminal and C-terminal domains is needed. In this review, we highlight studies supporting our current understanding of the structures and interactions of the N-terminal, globular, and C-terminal domains of linker histones within the nucleosome

CENP-A Nucleosome is a Sensitive Allosteric Scaffold for DNA and Chromatin Factors

Centromeric loci of chromosomes are defined by nucleosomes containing the histone H3 variant CENP-A, which bind their DNA termini more permissively than their canonical counterpart, a feature that is critical for the mitotic fidelity. A recent cryo-EM study demonstrated that the DNA termini of CENP-A nucleosomes, reconstituted with the Widom 601 DNA sequence, are asymmetrically flexible, meaning one terminus is more clearly resolved than the other. However, an earlier work claimed that both ends could be resolved in the presence of two stabilizing single chain variable fragment (scFv) antibodies per nucleosome, and thus are likely permanently bound to the histone octamer. This suggests that the binding of scFv antibodies to the histone octamer surface would be associated with CENP-A nucleosome conformational changes, including stable binding of the DNA termini. Here, we present computational evidence that allows to explain at atomistic level the structural rearrangements of CENP-A nucleosomes resulting from the antibody binding. The antibodies, while they only bind the octamer facades are capable of altering the dynamics of the nucleosomal core, and indirectly also the surrounding DNA. This effect has more drastic implications for the structure and the dynamics of the CENP-A nucleosome in comparison to its canonical counterpart. Furthermore, we find evidence that the antibodies bind the left and the right octamer facades at different affinities, another manifestation of the DNA sequence. We speculate that the cells could use induction of similar allosteric effects to control centromere function. (C) 2020 Elsevier Ltd. All rights reserved

Distinct Structures and Dynamics of Chromatosomes with Different Human Linker Histone Isoforms

The repeating structural unit of metazoan chromatin is the chromatosome, a nucleosome bound to a linker histone, H1 There are 11 human H1 isoforms with diverse cellular functions, but how they interact with the nucleosome remains elusive, Here, we determined the cryoelectron microscopy (cryo-EM) structures of chromatosomes containing 197 bp DNA and three different human H1 isoforms, respectively. The globular domains of all three H1 isoforms bound to the nucleosome dyad. However, the flanking/linker DNAs displayed substantial distinct dynamic conformations. Nuclear magnetic resonance (NMR) and H1 tail-swapping cryo-EM experiments revealed that the C-terminal tails of the H1 isoforms mainly controlled the flanking DNA orientations. We also observed partial ordering of the core histone H2A C-terminal and H3 N-terminal tails in the chromatosomes. Our results provide insights into the structures and dynamics of the chromatosomes and have implications for the structure and function of chromatin

Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle reveals differential flexibility of the DNA ends

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The CENP-A nucleosome: where and when it happens during the inner kinetochore's assembly

CENP-A is an essential histone variant that replaces the canonical H3 at the centromeres and marks these regions epigenetically. The CENP-A nucleosome is the specific building block of centromeric chromatin, and it is recognized by CENP-C and CENP-N, two components of the constitutive centromere-associated network (CCAN), the first protein layer of the kinetochore. Recent proposals of the yeast and human (h)CCAN structures position the assembly on exposed DNA, suggesting an elusive spatiotemporal recognition. We summarize the data on the structural organization of the CENP-A nucleosome and the binding of CENP-C and CENP-N. The latter posits an apparent contradiction in engaging the CENP-A nucleosome versus the CCAN. We propose a reconciliatory model for the assembly of CCAN on centromeric chromatin

Molecular Mechanism of Nucleosome Recognition by the Pioneer Transcription Factor Sox

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and physiochemistry approaches, along with DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific nucleosomal DNA recognition. Among three different nucleosome positions located on the positive DNA arm, a sequence-specific reading mechanism is solely satisfied at the superhelical location 2 (SHL2). While SHL2 acts transparently for solvent-facing Sox binding, among the other two positions, SHL4 permits only shape reading. The final position, SHL0 (dyad), on the other hand, allows no reading mechanism. These findings demonstrate that Sox-based nucleosome recognition is essentially guided by intrinsic nucleosome properties, permitting varying degrees of DNA recognition

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking