Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450_BM3 Variants

Cytochrome P450_BM3 is a heme-containing enzyme from <i>Bacillus megaterium</i> that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450_BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450_BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration <i>in vitro</i>. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex <i>in vivo</i>. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation

The hIKK2 X-ray crystal structure.

<p>(A) Domain organization schematics of full-length hIKK2 (above) and crystallized protein construct (below). The KD, ubiquitin-like domain (ULD), scaffold dimerization domain (SDD), and NEMO-binding region (N) are indicated and the domain borders are numbered. “EE” represents mutation of two active site serines 177 and 181 to glutamic acid residues in the crystallized protein. (B) Ribbon diagram representation of the hIKK2 X-ray crystal structure. Coloring and labels correspond to part A. Individual helices of the SDD are labeled as are the N and C termini (N and C, respectively). (C) Ribbon diagram representation of the hIKK2 KD with secondary structure elements and key amino acid side chain positions labeled. (D) Close-up view of the structural elements and amino acid residues immediately surrounding the activation loop (blue). (E) Superposition of hIKK2 (green) and xIKK2 (brown) KDs depicted in Cα-trace representation. Several amino acid residues that adopt significantly different positions in the two structures are rendered as sticks (yellow for hIKK2; orange for xIKK2) and labeled.</p

In vitro reconstitution of hIKK2 <i>trans</i> auto-phosphorylation.

<p>A catalytically inactive (D145N) and C-terminally truncated IKK2 (lanes 1–6) and mixtures of that enzyme with a catalytically active full-length version (lanes 4–6) were incubated with Mg-ATP for the time periods indicated and then probed via Western blot with anti-phosphoSer181 antibody (above) or by SDS PAGE (below).</p

Oligomerization of hIKK2 dimers.

<p>(A) Ribbon diagram of the interaction between two neighboring hIKK2 dimers in the crystal. Their asymmetric association gives rise to two unique intersubunit interfaces. (B) Close-up view of residues that interact between the KDs at the V-shaped interface. (C) Additional residues that mediate V-shaped interface interactions between the ULD an SDD. (D) Close-up view of interacting residues within the anti-parallel interface. (E) In vitro kinase assay reveals that catalytic activity of hIKK2 with mutations that disrupt the V-shaped interface (lanes 3–5) is drastically reduced compared to wild-type protein (WT-lane 2). (F) In vitro kinase assays with the same WT mutant proteins in which activation loop serines are mutated to glutamate. (G) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the V-shaped interface mutants correlates with activation loop phosphorylation status. (H) In vitro kinase assays reveal the modest effects on hIKK2 catalytic activity of mutation at the antiparallel interface.</p

Oligomerization of hIKK2 dimers in the crystal.

<p>(A) Three successive asymmetric units, each composed of six protomers, are taken from the hIKK2 X-ray crystal structure and depicted with the center asymmetric unit (labeled A–F) and colored with surface rendering as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001581#pbio-1001581-g003" target="_blank">Figure 3A</a>. Within this arrangement can be found four unique but closely related versions of the same dimer of dimers. Beginning from the left, there are four protomers (D, E, and another D′ and E′ from the symmetry-related asymmetric unit) that assemble into a tetramer. These four polypeptide chains are depicted as opaque, while the remaining protomers are rendered as semitransparent. In the second depiction of the same assembly of three asymmetric units, chains B, C, D, and E are rendered opaque and reveal themselves to assemble with a similar tetrameric arrangement. Likewise, protomers A, B, C, and F in the third panel and A, F, and the symmetry-related A″ and F″ chains assemble into renditions of the same tetramer. (B) Close-up views of the four unique assemblies viewed perpendicular to their 2-fold rotation axes reveal their close similarity.</p

IKK2 oligomerization activation model.

<p>(A) The hIKK2 X-ray crystal structure in space filling representation viewed from three different angles. The four surfaces that mediate oligomerization in the X-ray crystal structure are colored purple (dimer interface), blue (antiparallel interface), orange (V-shaped interface), and green (KD–KD interface). (B) A structure-based model for IKK2 activation via <i>trans</i> auto-phosphorylation. IKK2 interconverts between its open and closed dimeric forms. The open dimer can further associate to form transient homooligomers, such as observed in the hIKK2 X-ray crystal structure. Phosphorylation of one IKK2 subunit by an upstream kinase activates the kinase activity of that subunit and, as a consequence of its propensity to assemble into higher order oligomers through it V-shaped and KD-KD interfaces, is rapidly amplified via <i>trans</i> auto-phosphorylation.</p

A Structural Basis for IκB Kinase 2 Activation Via Oligomerization-Dependent <i>Trans</i> Auto-Phosphorylation

<div><p>Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of <i>Xenopus</i> IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote <i>trans</i> auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via <i>trans</i> auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent <i>trans</i> auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.</p></div

Interaction between KDs of oligomeric hIKK2.

<p>(A) Within the crystal, neighboring tetrameric assemblies interact symmetrically such that they contact one another through their V-shaped interfaces and two KDs are positioned within close proximity to one another (dashed box). (B) The close packed KDs are positioned so that their activation loops (dashed box) rest directly over the active site of a neighbor. Orange spheres mark the Cα positions on V229 and H232. (C) Close-up view of the kinase activation loops (yellow and blue) with glutamic acid residues 177 and 181 mimicking activation loop serines and the catalytic base D145 labeled. (D) In vitro kinase assay on immunoprecipitated hIKK2 with mutations at key residues that mediate KD–KD interactions in the crystal (lanes 3,4) reveals their involvement in catalytic activity. (E) Mutation of activation loop serines 177 and 181 to glutamates restores activity of immunoprecipitated IKK2 in vitro. (F) Immunoblotting with anti-phospho-Ser177,181 antibody reveals that the decrease in catalytic activity observed in the KD–KD interface mutants correlates with decreased activation loop phosphorylation.</p

Data collection and refinement statistics

a<p>Data in parentheses are for highest resolution shell.</p>b<p>Reflections with |<i>F</i>_O|/σ<1.0 rejected.</p>c<p>Calculated against a cross-validation set of 3.8% of data selected at random prior to refinement.</p>d<p>Combines clashscore, rotamer, and Ramachandran evaluations in to a single score, normalized to the same scale as x-ray resolution <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001581#pbio.1001581-Chen1" target="_blank">[40]</a>.</p