Molecular Mechanism of Copper Regulation of Mitogen-Activated Protein Kinase (MEK1)

Mutated constitutively activated forms of the BRAF kinase have been associated with various cancers, such as melanoma and leukemia. One such common BRAF mutant, BRAFV600E is found in about 50% of melanomas. BRAF is part of the MAPK signaling cascade that contains the downstream target MEK1/2, which phosphorylates ERK1/2 and subsequently increases cell proliferation. While the majority of the therapeutic inhibitors of the MAPK pathway have been designed to target BRAF, some have also targeted MEK1/2 as well as other kinases within the pathway such as ERK1/2. Recently, MEK1 has been observed to interact with copper (Cu). This element and copper transporter 1 (CTR1) were found to be important for kinase activity and when absent, decreased the ability of BRAFV600E to signal and mediate tumorigenesis. In this study, work was done to further characterize the MEK1-Cu interaction through crystallography, and carried out associated biochemical and enzymatic studies in order to characterize the functional consequence of the interaction and its effects on downstream ERK phosphorylation. A crystal structure of a MEK1-Cu complex was obtained revealing a Cu binding site to a regulatory site of the kinase that would be predicted to inhibit kinase activity. This structural observation is supported by functional studies with bacterially produced recombinant homogeneously purified MEK1 demonstrating that Cu inhibits MEK1 kinase activity in vitro. Interestingly, crudely purified MEK1 did show Cu-mediated MEK1 activation suggesting another mode of Cu-mediated MEK1 activation that is yet to be characterized. These results suggest that MEK1 is a metal-sensitive enzyme and that targeting these metals may be exploited to inhibit MAPK signaling for therapy

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAFMEK- ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS

Structural and Biochemical Studies of Cell-Grown Cry11Ba Insecticidal Protein Crystals

Nature self-assembles protein structures for various functions, including: storage, protection, and fortification. These self-assemblies range from filaments to full three-dimensional crystals and are pervasive across the tree of life. They include the granules present in immune system cells, the packing of hormones in the pancreas, the storage of proteins in plants, carboxysomes, viruses, and cell-grown crystals in microbes. To accomplish this, a better understanding of how certain organisms are able to naturally self-assemble macromolecules and how to recreate these in living cells must be achieved. Bacillus thuringiensis subsp. israelenesis’ (Bti) crystalline inclusions are of exceptional interest, since they naturally package a single protein into a crystalline inclusion through a life cycle process called sporulation. Compared to classical macromolecular crystallography that takes a plethora of variables to exhaustion can still yield no crystals. The laborious process could be prevented; however, by better understanding Bt and these crystalline inclusions’ cellular self-assembly process. Cry11Ba is a protein packed into these crystalline (Cry) inclusions and found to be one of the most toxic pesticidal proteins. These crystals are then ingested by their host and switch from their packaged toxin crystal to their inactive protoxin at the high pH within their gut. My interests have been in elucidating the macromolecular structure from in vivo produced crystals, further understanding the ambiguous mode of action to gain better perspective for other δ-endotoxins, and probing the self-assembly of the crystalline inclusions in vivo throughout the sporulation process. I have studied Cry11Ba with structural analysis, solubility & toxicity assays, mutational studies, and imaging capabilities developed in cryo-EM to successfully in solve a de novo structure via in vivo crystalline inclusions, charting the pH sensitivity of the crystals, identifying key residues for stability and toxicity, analyzed the monomeric and multimeric particles in alkaline environments to understand Cry11Ba’s mode of action, and visualized previously unobserved sporulation stages for Bti

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan , respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo -grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

International audienceCry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals.

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control