Spectroscopic characterization of the copper(I)-thiolate cluster in the DNA-binding domain of yeast ACE1 transcription factor

AbstractA polypeptide containing the amino-terminal region of ACEI (residues 1–122; 122*), the activator of yeast Cu-methallothionein gene transcription, shows charge-transfer and metal-centered UV absorption bands, and orange luminescence which are characteristic of Cu-cysteinyl thiolate cluster structures. These spectral features are abolished by the Cu(1) complexing agents CN* and diethyldithiocarbamate or exposure to acid, but not by the Cu(II)chelator. EDTA. Binding of the polypeptide to its specific DNA recognition site, but not to calf-thymus double-stranded DNA, induces quenching of its Tyr and Cu-S cluster luminescence emission. The CD spectrum is characteristic of a tightly folded structure that may be organized around the Cu cluster

Redistribution of Flexibility in Stabilizing Antibody Fragment Mutants Follows Le Chatelier's Principle

Le Châtelier's principle is the cornerstone of our understanding of chemical equilibria. When a system at equilibrium undergoes a change in concentration or thermodynamic state (i.e., temperature, pressure, etc.), La Châtelier's principle states that an equilibrium shift will occur to offset the perturbation and a new equilibrium is established. We demonstrate that the effects of stabilizing mutations on the rigidity ⇔ flexibility equilibrium within the native state ensemble manifest themselves through enthalpy-entropy compensation as the protein structure adjusts to restore the global balance between the two. Specifically, we characterize the effects of mutation to single chain fragments of the anti-lymphotoxin-β receptor antibody using a computational Distance Constraint Model. Statistically significant changes in the distribution of both rigidity and flexibility within the molecular structure is typically observed, where the local perturbations often lead to distal shifts in flexibility and rigidity profiles. Nevertheless, the net gain or loss in flexibility of individual mutants can be skewed. Despite all mutants being exclusively stabilizing in this dataset, increased flexibility is slightly more common than increased rigidity. Mechanistically the redistribution of flexibility is largely controlled by changes in the H-bond network. For example, a stabilizing mutation can induce an increase in rigidity locally due to the formation of new H-bonds, and simultaneously break H-bonds elsewhere leading to increased flexibility distant from the mutation site via Le Châtelier. Increased flexibility within the VH β4/β5 loop is a noteworthy illustration of this long-range effect

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

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

Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

<div><p>The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.</p></div

Molecular dynamics trajectories.

<p>FA, CA and EA represent anti-fluorescein, Anti-CD3 and esterase catalytic antibodies, respectively. (A) Root mean square deviations (RMSDs) of Cα are provided each of the molecular dynamics trajectories. The FA(GL) and FA(AM) exhibit larger RMSDs than other antibodies due to change of domain-domain reorientation. (B) Global RMSDs of for the full FA(GL) Fab and individual RMSDs for each domain. All the four domains (VH, VL, CH and CL) show much lower RMSDs than global RMSDs. The small fluctuations within the domains highlight that the global fluctuations are caused by slippage along the domain interface, where the four domains are continually rearranging relative to each other. (C) The slippage along the domain interfaces is indicated in panel (B), where different colors represent snapshots occurring at: 20 ns (red), 40 ns (blue), and 80 ns (green).</p

Statistically significant changes in rigidity (p<0.01) are tabulated based on the distance between them and the closest constituent mutation.

<p>Note that the average distance in the increased rigidity response (A) is significantly less than increased flexibility (B), 13.6 Å vs. 17.9 Å, respectively.</p