Tom Huxford

Structures of Aplysia Achbp Complexes with Nicotinic Agonists and Antagonists Reveal Distinctive Binding Interfaces and Conformations.

Upon ligand binding at the subunit interfaces, the extracellular domain of the nicotinic acetylcholine receptor undergoes conformational changes, and agonist binding allosterically triggers opening of the ion channel. The soluble acetylcholine‐binding protein (AChBP) from snail has been shown to be a structural and functional surrogate of the ligand‐binding domain (LBD) of the receptor. Yet, individual AChBP species display disparate affinities for nicotinic ligands. The crystal structure of AChBP from Aplysia californica in the apo form reveals a more open loop C and distinctive positions for other surface loops, compared with previous structures. Analysis of Aplysia AChBP complexes with nicotinic ligands shows that loop C, which does not significantly change conformation upon binding of the antagonist, methyllycaconitine, further opens to accommodate the peptidic antagonist, α‐conotoxin ImI, but wraps around the agonists lobeline and epibatidine. The structures also reveal extended and nonoverlapping interaction surfaces for the two antagonists, outside the binding loci for agonists. This comprehensive set of structures reflects a dynamic template for delineating further conformational changes of the LBD of the nicotinic receptor.

The Crystal Structure of the IκBα/NF-κB Complex Reveals Mechanisms of NF-κB Inactivation

IkappaBalpha regulates the transcription factor NF-kappaB through the formation of stable IkappaBalpha/NF-kappaB complexes. Prior to induction, IkappaBalpha retains NF-kappaB in the cytoplasm until the NF-kappaB activation signal is received. After activation, NF-kappaB is removed from gene promoters through association with nuclear IkappaBalpha, restoring the preinduction state. The 2.3 A crystal structure of IkappaBalpha in complex with the NF-kappaB p50/p65 heterodimer reveals mechanisms of these inhibitory activities. The presence of IkappaBalpha allows large en bloc movement of the NF-kappaB p65 subunit amino-terminal domain. This conformational change induces allosteric inhibition of NF-kappaB DNA binding. Amino acid residues immediately preceding the nuclear localization signals of both NF-kappaB p50 and p65 subunits are tethered to the IkappaBalpha amino-terminal ankyrin repeats, impeding NF-kappaB from nuclear import machinery recognition.

IκBα Functions through Direct Contacts with the Nuclear Localization Signals and the DNA Binding Sequences of NF-κB

We have determined the binding energies of complexes formed between IκBα and the wild type and mutational variants of three different Rel/NF-κB dimers, namely, the p50/p65 heterodimer and homodimers of p50 and p65. We show that although a common mode of interaction exists between the Rel/NF-κB dimers and IκBα, IκBα binds the NF-κB p50/p65 heterodimer with 60- and 27-fold higher affinity than the p50 and p65 homodimers, respectively. Each of the three flexibly linked segments of the rel homology region of Rel/NF-κB proteins (the nuclear localization sequence, the dimerization domain, and the amino-terminal DNA binding domain) is directly engaged in forming the protein/protein interface with the ankyrin repeats and the carboxyl-terminal acidic tail/PEST sequence of IκBα. In the cell, IκBα functions to retain NF-κB in the cytoplasm and inhibit its DNA binding activity. These properties are a result of the direct involvement of the nuclear localization sequences and of the DNA binding region of NF-κB in complex with IκBα. A model of the interactions in the complex is proposed based on our observations and the crystal structures of Rel/NF-κB dimers and the ankyrin domains of related proteins.

Biophysical characterization of the free IκBα ankyrin repeat domain in solution

The crystal structure of IκBα in complex with the transcription factor, nuclear factor κ‐B (NF‐κB) shows six ankyrin repeats, which are all ordered. Electron density was not observed for most of the residues within the PEST sequence, although it is required for high‐affinity binding. To characterize the folded state of IκBα (67–317) when it is not in complex with NF‐κB, we have carried out circular dichroism (CD) spectroscopy, 8‐anilino‐1‐napthalenesulphonic acid (ANS) binding, differential scanning calorimetry, and amide hydrogen/deuterium exchange experiments. The CD spectrum shows the presence of helical structure, consistent with other ankyrin repeat proteins. The large amount of ANS‐binding and amide exchange suggest that the protein may have molten globule character. The amide exchange experiments show that the third ankyrin repeat is the most compact, the second and fourth repeats are somewhat less compact, and the first and sixth repeats are solvent exposed. The PEST extension is also highly solvent accessible. Iκ Bα unfolds with a Tm of 42°C, and forms a soluble aggregate that sequesters helical and variable loop parts of the first, fourth, and sixth repeats and the PEST extension. The second and third repeats, which conform most closely to a consensus for stable ankyrin repeats, appear to remain outside of the aggregate. The ramifications of these observations for the biological function of IκBα are discussed.

Mechanism of IκBα Binding to NF-κB Dimers

X-ray crystal structures of the NF-κB·IκBα complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both IκBα and NF-κB. In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity. IκBα preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker. Our results indicate that the nuclear localization sequence and the region C-terminal to it of the NF-κB p65 subunit is a major contributor to NF-κB·IκBα complex formation. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and IκBα. A second set of interactions involving the acidic C-terminal/PEST-like region of IκBα and the NF-κB p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking. Phosphorylation of the C-terminal/PEST-like region by casein kinase II further enhances binding.

Mechanism of IkappaBalpha binding to NF-kappaB dimers

X-ray crystal structures of the NF-κB·IκBα complex revealed an extensive and complex protein-protein interface involving independent structural elements present in both IκBα and NF-κB. In this study, we employ a gel electrophoretic mobility shift assay to assess and quantitate the relative contributions of the observed interactions toward overall complex binding affinity. IκBα preferentially binds to the p50/p65 heterodimer and p65 homodimer, with binding to p50 homodimer being significantly weaker. Our results indicate that the nuclear localization sequence and the region C-terminal to it of the NF-κB p65 subunit is a major contributor to NF-κB·IκBα complex formation. Additionally, there are no contacts between the corresponding nuclear localization signal tetrapeptide of p50 and IκBα. A second set of interactions involving the acidic C-terminal/PEST-like region of IκBα and the NF-κB p65 subunit N-terminal domain also contributes binding energy toward formation of the complex. This interaction is highly dynamic and nonspecific in nature, as shown by oxidative cysteine cross-linking. Phosphorylation of the C-terminal/PEST-like region by casein kinase II further enhances binding.

The role of DNA in the mechanism of NFκB dimer formation: crystal structures of the dimerization domains of the p50 and p65 subunits

BACKGROUND Members of the rel/NFkappaB family of transcription factors play a vital role in the regulation of rapid cellular responses, such as those required to fight infection or react to cellular stress. Members of this family of proteins form homo- and heterodimers with differing affinities for dimerization. They share a structural motif known as the rel homology region (RHR), the C-terminal one third of which mediates protein dimerization. Crystal structures of the rel/NFkappaB family members p50 and p65 in their DNA-bound homodimeric form have been solved. These structures showed that the residues from the dimerization domains of both p50 and p65 participate in DNA binding and that the DNA-protein and protein dimerization surfaces form one continuous overlapping interface. We desired to investigate the contribution of DNA to NFkappaB dimerization and to identify the mechanism for the selective association of rel/NFkappaB family peptides into transcriptionally active dimers. RESULTS We report here the crystal structures of the dimerization domains of murine p50 and p65 at 2.2 A and 2.0 A resolution, respectively. A comparison of these two structures suggests that conservative amino acid changes at three positions are responsible for the differences in their dimer interfaces. The presence of the target DNA does not change the dimer interface of either protein in any significant manner. CONCLUSIONS These two structures suggest that the rel/NFkappaB family of transcription factors use only a few conservative changes in their amino acid sequences to form a host of dimers with varying affinities for dimerization. Amino acids at positions corresponding to 254, 267, and 307 of murine p50, function as primary determinants for the observed differences in dimerization affinity. The DNA-contacting charged amino acid sidechains from the dimerization domains are held in a similar conformation in both the DNA-bound and free states, therefore, no major structural rearrangement is required to bring these residues into contact with the DNA.

A Structural Guide to Proteins of the NF-κB Signaling Module

The prosurvival transcription factor NF-kappaB specifically binds promoter DNA to activate target gene expression. NF-kappaB is regulated through interactions with IkappaB inhibitor proteins. Active proteolysis of these IkappaB proteins is, in turn, under the control of the IkappaB kinase complex (IKK). Together, these three molecules form the NF-kappaB signaling module. Studies aimed at characterizing the molecular mechanisms of NF-kappaB, IkappaB, and IKK in terms of their three-dimensional structures have lead to a greater understanding of this vital transcription factor system.

A Structural Basis for IκB Kinase 2 Activation Via Oligomerization-Dependent Trans Auto-Phosphorylation

Conformational change in human IKK2 permits dimers to form higher-order oligomers that support interaction between kinase domains and promote activation through trans auto-phosphorylation.

Structural characterization of agonist and antagonist-bound acetylcholine-binding protein from Aplysia californica.

Nicotinic acetylcholine receptors (nAChRs) are well-characterized allosteric transmembrane proteins involved in the rapid gating of ions elicited by ACh. These receptors belong to the Cys-loop superfamily of ligand-gated ion channels, which also includes GABAA and GABAC, 5-HT3, and glycine receptors. The nAChRs are homo- or heteromeric pentamers of structurally related subunits that encompass an extracellular N-terminal ligand-binding domain, four transmembrane-spanning regions that form the ion channel, and an extended intracellular region between spans 3 and 4. Ligand binding triggers conformational changes that are transmitted to the transmembrane-spanning region, leading to gating and changes in membrane potential. The four transmembrane spans on each of the five subunits create a substantial region of hydrophobicity that precludes facile crystallization of this protein. However the freshwater snail, Lymnaea stagnalis, produces a soluble homopentameric protein, termed the ACh-binding protein (AChBP), which binds ACh (Smit et al., 2001). Its structure was determined recently (Brejc et al., 2001) at high resolution, revealing the structural scaffold for nAChR, and has become a functional and structural surrogate of the nAChR ligand-binding domain. We have characterized an AChBP from Aplysia californica and determined distinct ligand-binding properties when compared to those of L. stagnalis, including ligand specificity for the nAChR alpha7 subtype-specific alpha-conotoxin ImI (Hansen et al., 2004).