Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Châtelier’s Principle

Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas‐Finet, José R.; Jacobs, Donald J.; Livesay, Dennis R.

doi:10.1371/journal.pone.0092870

Cited by 40 publications

(58 citation statements)

References 67 publications

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“…Despite added torsion constraints to the Mos1 and SB structures, locally increasing rigidity along the backbone, the difference in backbone rigidity between DNA‐bound and DNA‐unbound states of Mos1 and SB transposases demonstrates a rich distribution of regions with increased and decreased rigidity (Figure ). This happens because of the enthalpy/entropy compensation due to global changes in flexibility/rigidity as was previously observed as a general effect along the lines of a le Chatelier principle . As expected, we find that there is a dramatic loss of flexibility in the clamp loop that forms multiple protein‐protein and protein‐DNA contacts .…”

Section: Resultssupporting

confidence: 85%

Rigidity and flexibility characteristics of DD[E/D]‐transposases Mos1 and Sleeping Beauty

et al. 2019

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DD[E/D]-transposases catalyze the multistep reaction of cut-and-paste DNA transposition. Structurally, several DD[E/D]-transposases have been characterized, revealing a multi-domain structure with the catalytic domain possessing the RNase H-like structural motif that brings three catalytic residues (D, D, and E or D) into close proximity for the catalysis. However, the dynamic behavior of DD[E/D]-transposases during transposition remains poorly understood. Here, we analyze the rigidity and flexibility characteristics of two representative DD[E/D]-transposases Mos1 and Sleeping Beauty (SB) using the minimal distance constraint model (mDCM). We find that the catalytic domain of both transposases is globally rigid, with the notable exception of the clamp loop being flexible in the DNA-unbound form. Within this globally rigid structure, the central β-sheet of the RNase H-like motif is much less rigid in comparison to its surrounding α-helices, forming a cage-like structure. The comparison of the original SB transposase to its hyperactive version SB100X reveals the region where the change in flexibility/rigidity correlates with increased activity. This region is found to be within the RNase H-like structural motif and comprise the loop leading from beta-strand B3 to helix H1, helices H1 and H2, which are located on the same side of the central beta-sheet, and the loop between helix H3 and beta-strand B5. We further identify the RKEN214-217DAVQ mutations of the set of hyperactive mutations within the catalytic domain of SB transposase to be the driving factor that induces change in residue-pair rigidity correlations within SB transposase. Given that a signature RNase H-like structural motif is found in DD[E/D]-transposases and, more broadly, in a large superfamily of polynucleotidyl transferases, our results are relevant to these proteins as well. K E Y W O R D S distance constraint model, DNA transposon, dynamics, flexibility and rigidity, fluorescence, protein-DNA complex, transposase that includes enzymes involved in replication, recombination, DNA repair, splicing, transposition, RNA interference (RNAi), and CRISPR-Cas immunity. 15,16 The RNase H-like structural motif is schematically shown in Figure 1. It consists of a central β-sheet comprised of five β-strands (ordered 32 145 with the second β-strand B2 antiparallel to other strands) and three surrounding α-helices. Two of the three α-helices (H1 and H2) are inserted in the amino acid sequence between β-strands B3 and B4 and B4 and B5, respectively, and are located on one side of the β-sheet. The third α-helix (H3) is located on the opposite side of the β-sheet and either immediately follows β-strand B5 or is separated from it by inserted amino acid sequence of various length. The most abundant proteins in the RNHL superfamily are DD[E/D]-transposases. 15 Although there are significant differences in sequence and structure across the RNHL superfamily, the RNase H architecture represents a highly conserved core of the catalytic domain of RNHL proteins, suggesting...

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Section: Resultssupporting

confidence: 85%

Rigidity and flexibility characteristics of DD[E/D]‐transposases Mos1 and Sleeping Beauty

et al. 2019

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“…This trend is consistent with a previous finding that highly localized rigidifying perturbations induce flexibility at distal locations in similar spirit to the Le Chatelier’s principle. 9 …”

Section: Resultsmentioning

confidence: 99%

“…9, 26 Additionally’ one representative conformation was selected from each MD trajectory within the most and least populated clusters based on RMSD clustering analysis. Starting from these two structures the same protocols were followed to run 100 ns MD simulations using the GROMACS 5.1.2 package 48 resulting in three independent MD trajectories for native scFv and for each of the 5 mutants.…”

Section: Methodsmentioning

confidence: 99%

Decomposing Dynamical Couplings in Mutated scFv Antibody Fragments into Stabilizing and Destabilizing Effects

Ramaprasad

Uddin²,

Casas‐Finet³

et al. 2017

J. Am. Chem. Soc.

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Conformational fluctuations within scFv antibodies are characterized by a novel perturbation-response decomposition of molecular dynamics trajectories. Both perturbation and response profiles are stratified into stabilizing and destabilizing conditions. The linker between the VH and VL domains exhibits the dominant dynamical response by being coupled to nearly the entire protein, responding to both stabilizing and destabilizing perturbations. Perturbations within complementarity-determining regions (CDR) induce rich behavior in dynamic response. Among many effects, stabilizing any CDR loop in the VH domain triggers a destabilizing response in all CDR loops in the VL domain and vice versa. Destabilizing residues within the VL domain are likely to stabilize all CDR loops in the VH domain, and, when these residues are not buried the CDR loops in the VL domain are also likely to be stabilized. These effects, described by shifts in normal mode characteristics, initiate a propensity for dynamic allostery with possible functional implications in bispecific antibodies.

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“…However, the apparent destabilization of the protein detected by a lowered T m may result from entropic compensation elsewhere in the protein framework. This situation has been described as a rigidity-flexibility equilibrium, where lowered entropy due to a stabilizing mutation at one site is compensated by increased flexibility in remote regions (39) . The reverse can also happen when the mutation is destabilizing.…”

Section: Discussionmentioning

confidence: 99%

Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness

et al. 2017

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Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin-resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upwards of 60 amino acid changes relative to wild-type PBP2, or by non-mosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates were identified that harbored a mosaic penA allele with an A501P mutation. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon-501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, A501S) were isolated that increased resistance and preserved essential transpeptidase function. To understand their structural implications, these mutations were introduced into the non-mosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was solved and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active-site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

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Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Châtelier’s Principle

Cited by 40 publications

References 67 publications

Rigidity and flexibility characteristics of DD[E/D]‐transposases Mos1 and Sleeping Beauty

Rigidity and flexibility characteristics of DD[E/D]‐transposases Mos1 and Sleeping Beauty

Decomposing Dynamical Couplings in Mutated scFv Antibody Fragments into Stabilizing and Destabilizing Effects

Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness

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