16 research outputs found
Tradeoff Between Stability and Multispecificity in the Design of Promiscuous Proteins
Natural proteins often partake in several highly specific protein-protein interactions. They are thus subject to multiple opposing forces during evolutionary selection. To be functional, such multispecific proteins need to be stable in complex with each interaction partner, and, at the same time, to maintain affinity toward all partners. How is this multispecificity acquired through natural evolution? To answer this compelling question, we study a prototypical multispecific protein, calmodulin (CaM), which has evolved to interact with hundreds of target proteins. Starting from high-resolution structures of sixteen CaM-target complexes, we employ state-of-the-art computational methods to predict a hundred CaM sequences best suited for interaction with each individual CaM target. Then, we design CaM sequences most compatible with each possible combination of two, three, and all sixteen targets simultaneously, producing almost 70,000 low energy CaM sequences. By comparing these sequences and their energies, we gain insight into how nature has managed to find the compromise between the need for favorable interaction energies and the need for multispecificity. We observe that designing for more partners simultaneously yields CaM sequences that better match natural sequence profiles, thus emphasizing the importance of such strategies in nature. Furthermore, we show that the CaM binding interface can be nicely partitioned into positions that are critical for the affinity of all CaM-target complexes and those that are molded to provide interaction specificity. We reveal several basic categories of sequence-level tradeoffs that enable the compromise necessary for the promiscuity of this protein. We also thoroughly quantify the tradeoff between interaction energetics and multispecificity and find that facilitating seemingly competing interactions requires only a small deviation from optimal energies. We conclude that multispecific proteins have been subjected to a rigorous optimization process that has fine-tuned their sequences for interactions with a precise set of targets, thus conferring their multiple cellular functions
Computational Comparison Studies of Quadratic Assignment Like Formulations for the In Silico Sequence Selection Problem in De Novo Protein Design
Second-Sphere Interactions between the C93–Y157 Cross-Link and the Substrate-Bound Fe Site Influence the O<sub>2</sub> Coupling Efficiency in Mouse Cysteine Dioxygenase
Cysteine dioxygenase (CDO) is a non-heme
iron enzyme that catalyzes
the O<sub>2</sub>-dependent oxidation of l-cysteine (l-Cys) to produce cysteinesulfinic acid (CSA). Adjacent to the
Fe site of CDO is a covalently cross-linked cysteine–tyrosine
pair (C93–Y157). While several theories have been proposed
for the function of the C93–Y157 pair, the role of this post-translational
modification remains unclear. In this work, the steady-state kinetics
and O<sub>2</sub>/CSA coupling efficiency were measured for wild-type
CDO and selected active site variants (Y157F, C93A, and H155A) to
probe the influence of second-sphere enzyme–substrate interactions
on catalysis. In these experiments, it was observed that both <i>k</i><sub>cat</sub> and the O<sub>2</sub>/CSA coupling efficiency
were highly sensitive to the presence of the C93–Y157 cross-link
and its proximity to the substrate carboxylate group. Complementary
electron paramagnetic resonance (EPR) experiments were performed to
obtain a more detailed understanding of the second-sphere interactions
identified in O<sub>2</sub>/CSA coupling experiments. Samples of the
catalytically inactive substrate-bound Fe<sup>III</sup>–CDO
species were treated with cyanide, resulting in a low-spin (<i>S</i> = <sup>1</sup>/<sub>2</sub>) ternary complex. Remarkably,
both the presence of the C93–Y157 pair and interactions with
the Cys carboxylate group could be readily identified by perturbations
to the rhombic EPR signal. Spectroscopically validated active site
quantum mechanics/molecular mechanics and density functional theory
computational models are provided to suggest a potential role for
Y157 in the positioning of the substrate Cys in the active site and
to verify the orientation of the <b>g</b>-tensor relative to
the CDO Fe site molecular axis