Gourisankar Ghosh

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.

Transcriptional regulation via the NF-κB signaling module

Stimulus-induced nuclear factor-κB (NF-κB) activity, the central mediator of inflammatory responses and immune function, comprises a family of dimeric transcription factors that regulate diverse gene expression programs consisting of hundreds of genes. A family of inhibitor of κB (IκB) proteins controls NF-κB DNA-binding activity and nuclear localization. IκB protein metabolism is intricately regulated through stimulus-induced degradation and feedback re-synthesis, which allows for dynamic control of NF-κB activity. This network of interactions has been termed the NF-κB signaling module. Here, we summarize the current understanding of the molecular structures and biochemical mechanisms that determine NF-κB dimer formation and the signal-processing characteristics of the signaling module. We identify NF-κB–κB site interaction specificities and dynamic control of NF-κB activity as mechanisms that generate specificity in transcriptional regulation. We discuss examples of gene regulation that illustrate how these mechanisms may interface with other transcription regulators and promoter-associated events, and how these mechanisms suggest regulatory principles for NF-κB-mediated gene activation.

Crystal structure of p50/p65 heterodimer of transcription factor NF-kappaB bound to DNA.

The NF-κB p50/p65 heterodimer is the classical member of the Relfamily of transcription factors which regulate diverse cellular functions such as immune response, cell growth, and development. Other mammalian Rel family members, including theproteins p52, proto-oncoprotein c-Rel, and RelB, all have amino-terminal Rel-homology regions (RHRs). The RHR is responsible for the dimerization, DNA binding and cytosolic localization of these proteins by virtue of complex formation with inhibitor κB proteins. Signal-induced removal of κB inhibitors allows translocation of dimers to the cell nucleus and transcriptional regulation of κB DNA-containing genes. NF-κB specifically recognizes κB DNA elements,, with a consensus sequence of 5′-GGGRNYYYCC-3′ (R is an unspecified purine; Y is an unspecified pyrimidine; and N is any nucleotide). Here we report the crystal structure at 2.9 Å resolution of the p50/p65 heterodimer bound to the κB DNA of the intronic enhancer of the immunoglobulin light-chain gene. Our structure reveals a 5-base-pair 5′ subsite for p50, and a 4-base-pair 3′ subsite for p65. This structure indicates why the p50/p65 heterodimer interface is stronger than that of either homodimer. A comparison of this structure with those of other Rel dimers reveals that both subunits adopt variable conformations in a DNA-sequence-dependent manner. Our results explain the different behaviour of the p50/p65 heterodimer with heterologous promoters.

A Fourth IκB Protein within the NF-κB Signaling Module

An assay method incorporating at least two different chemiluminescent compounds for detection and/or quantitation of at least two substances in a test sample is described. The synthesis of chemiluminescent reagents or conjugates for use in such methods as well as kits incorporating such reagents are also disclosed. The assays have particular application in the field of clinical diagnostics.

Regulation of DNA binding by Rel/NF-κB transcription factors : structural views

Rel/NF-κB transcription factors form homo- and heterodimers with different DNA binding site specificities and DNA binding affinities. Several intracellular pathways evoked by a wide range of biological factors and environmental conditions can lead to the activation of Rel/NF-κB dimers by signaling degradation of the inhibitory IκB protein. In the nucleus Rel/NF-κB dimers modulate the expression of a variety of genes including those encoding cytokines, growth factors, acute phase response proteins, immunoreceptors, other transcription factors, cell adhesion molecules, viral proteins and regulators of apoptosis. The primary focus of this review is on structural and functional aspects of Rel/NF-κB:DNA complexes and their formation. The salient features of the Rel/NF-κB dimer:DNA structure are described, as are modes of transcriptional regulation by phosphorylation, altered DNA binding properties, varying protein conformations, and interactions with IκB proteins.

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.

Crystal structure of NF-κB (p50)2 complexed to a high-affinity RNA aptamer

We have recently identified an RNA aptamer for the transcription factor NF-κB p50 homodimer [(p50)2], which exhibits little sequence resemblance to the consensus DNA target for (p50)2, but binds (p50)2 with an affinity similar to that of the optimal DNA target. We describe here the 2.45-Å resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem–loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3′ helix, together with the major groove of the irregular 3′ helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the κB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50)2.

NF‐κB dictates the degradation pathway of IκBα

IκB proteins are known as the regulators of NF‐κB activity. They bind tightly to NF‐κB dimers, until stimulus‐responsive N‐terminal phosphorylation by IKK triggers their ubiquitination and proteasomal degradation. It is known that IκBα is an unstable protein whose rapid degradation is slowed upon binding to NF‐κB, but it is not known what dynamic mechanisms control the steady‐state level of total IκBα. Here, we show clearly that two degradation pathways control the level of IκBα. Free IκBα degradation is not controlled by IKK or ubiquitination but intrinsically, by the C‐terminal sequence known as the PEST domain. NF‐κB binding to IκBα masks the PEST domain from proteasomal recognition, precluding ubiquitin‐independent degradation; bound IκBα then requires IKK phosphorylation and ubiquitination for slow basal degradation. We show the biological requirement for the fast degradation of the free IκBα protein; alteration of free IκBα degradation dampens NF‐κB activation. In addition, we find that both free and bound IκBα are similar substrates for IKK, and the preferential phosphorylation of NF‐κB‐bound IκBα is due to stabilization of IκBα by NF‐κB.

Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones.

Phosphorylation is essential for the SR family of splicing factors/regulators to function in constitutive and regulated pre-mRNA splicing; yet both hypo- and hyperphosphorylation of SR proteins are known to inhibit splicing, indicating that SR protein phosphorylation must be tightly regulated in the cell. However, little is known how SR protein phosphorylation might be regulated during development or in response to specific signaling events. Here, we report that SRPK1, a ubiquitously expressed SR protein-specific kinase, directly binds to the cochaperones Hsp40/DNAjc8 and Aha1, which mediate dynamic interactions of the kinase with the major molecular chaperones Hsp70 and Hsp90 in mammalian cells. Inhibition of the Hsp90 ATPase activity induces dissociation of SRPK1 from the chaperone complexes, which can also be triggered by a stress signal (osmotic shock), resulting in translocation of the kinase from the cytoplasm to the nucleus, differential phosphorylation of SR proteins, and alteration of splice site selection. These findings connect the SRPK to the molecular chaperone system that has been implicated in numerous signal transduction pathways and provide mechanistic insights into complex regulation of SR protein phosphorylation and alternative splicing in response to developmental cues and cellular signaling.

Mechanism of κB DNA binding by Rel/NF-κB dimers

The DNA binding of three different NF-κB dimers, the p50 and p65 homodimers and the p50/p65 heterodimer, has been examined using a combination of gel mobility shift and fluorescence anisotropy assays. The NF-κB p50/p65 heterodimer is shown here to bind the κB DNA target site of the immunoglobulin κ enhancer (Ig-κB) with an affinity of approximately 10 nm. The p50 and p65 homodimers bind to the same site with roughly 5- and 15-fold lower affinity, respectively. The nature of the binding isotherms indicates a cooperative mode of binding for all three dimers to the DNA targets. We have further characterized the role of pH, salt, and temperature on the formation of the p50/p65 heterodimer-Ig-κB complex. The heterodimer binds to the Ig-κB DNA target in a pH-dependent manner, with the highest affinity between pH 7.0 and 7.5. A strong salt-dependent interaction between Ig-κB and the p50/p65 heterodimer is observed, with optimum binding occurring at monovalent salt concentrations below 75 mm, with binding becoming virtually nonspecific at a salt concentration of 200 mm. Binding of the heterodimer to DNA was unchanged across a temperature range between 4 °C and 42 °C. The sensitivity to ionic environment and insensitivity to temperature indicate that NF-κB p50/p65 heterodimers form complexes with specific DNA in an entropically driven manner.