De-Bin Huang

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.

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.

NF-κB regulation: lessons from structures.

Summary:  The signaling module that specifies nuclear factor‐κΒ (NF‐κB) activation is a three‐component system: NF‐κB, inhibitor of NF‐κΒ (IκΒ), and IκΒ kinase complex (IKK). IKK receives upstream signals from the surface or inside the cell and converts itself into a catalytically active form, leading to the destruction of IκB in the inhibited IκB:NF‐κB complex, leaving active NF‐κB free to regulate target genes. Hidden within this simple module are family members that all can undergo various modifications resulting in expansion of functional spectrum. Three‐dimensional structures representing all three components are now available. These structures have allowed us to interpret cellular observations in molecular terms and at the same time helped us to bring forward new concepts focused towards understanding the specificity in the NF‐κB activation pathway.

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 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.

NF-κB p52:RelB heterodimer recognizes two classes of κB sites with two distinct modes

The X‐ray structure of the nuclear factor‐κB (NF‐κB) p52:RelB:κB DNA complex reveals a new recognition feature not previously seen in other NF‐κB:κB DNA complexes. Arg 125 of RelB is in contact with an additional DNA base pair. Surprisingly, the p52:RelB R125A mutant heterodimer shows defects both in DNA binding and in transcriptional activity only to a subclass of κB sites. We found that the Arg 125‐sensitive κB sites contain more contiguous and centrally located A:T base pairs than do the insensitive sites. A protein‐induced kink observed in this complex, which used an AT‐rich κB site, might allow the DNA contact by Arg 125; such a kink might not be possible in complexes with non‐AT‐rich κB sites. Furthermore, we show that the p52:RelB heterodimer binds to a broader spectrum of κB sites when compared with the p50:RelA heterodimer. We suggest that the p52:RelB heterodimer is more adaptable to complement sequence and structural variations in κB sites when compared with other NF‐κB dimers.

p100/IκBδ sequesters and inhibits NF-κB through kappaBsome formation

Significance NF-κB activation pathways program cells to induce various activities including survival and inflammation. NF-κB activation requires inhibitor IκB degradation. Two of the IκB proteins, p100 and p105, are also the precursors of two NF-κB proteins, p52 and p50. The mechanism by which the p100 inhibits NF-κB remains elusive. The X-ray structure of the C-terminal domain of p100 reveals tetrameric organization and an essential function of tetramerization in sequestering four NF-κB molecules in a subunit-independent manner. Such broader nonspecific NF-κB inhibition by p100 contrasts with the functions of other IκB proteins. A host of genetic mutations linked to excessive p100 processing derail homeostatic NF-κB activity. Our work provides a molecular framework to understand homeostatic regulation of p100 function. Degradation of I kappaB (κB) inhibitors is critical to activation of dimeric transcription factors of the NF-κB family. There are two types of IκB inhibitors: the prototypical IκBs (IκBα, IκBβ, and IκBε), which form low-molecular-weight (MW) IκB:NF-κB complexes that are highly stable, and the precursor IκBs (p105/IκBγ and p100/IκBδ), which form high-MW assemblies, thereby suppressing the activity of nearly half the cellular NF-κB [Savinova OV, Hoffmann A, Ghosh G (2009) Mol Cell 34(5):591–602]. The identity of these larger assemblies and their distinct roles in NF-κB inhibition are unknown. Using the X-ray crystal structure of the C-terminal domain of p100/IκBδ and functional analysis of structure-guided mutants, we show that p100/IκBδ forms high-MW (IκBδ)4:(NF-κB)4 complexes, referred to as kappaBsomes. These IκBδ-centric “kappaBsomes” are distinct from the 2:2 complexes formed by IκBγ. The stability of the IκBδ tetramer is enhanced upon association with NF-κB, and hence the high-MW assembly is essential for NF-κB inhibition. Furthermore, weakening of the IκBδ tetramer impairs both its association with NF-κB subunits and stimulus-dependent processing into p52. The unique ability of p100/IκBδ to stably interact with all NF-κB subunits by forming kappaBsomes demonstrates its importance in sequestering NF-κB subunits and releasing them as dictated by specific stimuli for developmental programs.

Structural Basis for the Activation of IKK1/α

Summary Distinct signaling pathways activate the NF-κB family of transcription factors. The canonical NF-κB-signaling pathway is mediated by IκB kinase 2/β (IKK2/β), while the non-canonical pathway depends on IKK1/α. The structural and biochemical bases for distinct signaling by these otherwise highly similar IKKs are unclear. We report single-particle cryoelectron microscopy (cryo-EM) and X-ray crystal structures of human IKK1 in dimeric (∼150 kDa) and hexameric (∼450 kDa) forms. The hexamer, which is the representative form in the crystal but comprises only ∼2% of the particles in solution by cryo-EM, is atrimer of IKK1 dimers. While IKK1 hexamers are not detectable in cells, the surface that supports hexamer formation is critical for IKK1-dependent cellular processing of p100 to p52, the hallmark of non-canonical NF-κB signaling. Comparison of this surface to that in IKK2 indicates significant divergence, and it suggests a fundamental role for this surface in signaling by these kinases through distinct pathways.

The SRSF1 linker induces semi-conservative ESE binding by cooperating with the RRMs

SR proteins promote spliceosome formation by recognizing exonic splicing enhancers (ESEs) during pre-mRNA splicing. Each SR protein binds diverse ESEs using strategies that are yet to be elucidated. Here, we show that the RNA-binding domain (RBD) of SRSF1 optimally binds to decameric purine rich ESE sequences although locations of purines are not stringently specified. The presence of uracils either within or outside of the recognition site is detrimental for binding with SRSF1. The entire RBD, comprised of two RRMs and a glycine-rich linker, is essential for ESE binding. Mutation within each segment reduced or nearly abolished binding, suggesting that these segments mediate cooperative binding. The linker plays a decisive role in organizing ESE binding. The flanking basic regions of the linker appear to communicate with each other in bringing the two RRMs close together to form the complex with RNA. Our study thus suggests semi-conservative adaptable interaction between ESE and SRSF1, and such binding mode is not only essential for the recognition of plethora of physiological ESE sequences but may also be essential for the interaction with various factors during the spliceosome assembly.

A Structural Basis for Selective Dimerization by NF-κB RelB

Transcription factors of the nuclear factor kappaB (NF-κB) family arise through the combinatorial association of five distinct Rel subunits into functional dimers. However, not every dimer combination is observed in cells. The RelB subunit, for example, does not appear as a homodimer and forms heterodimers exclusively in combination with p50 or p52 subunits. We previously reported that the RelB homodimer could be forced to assemble through domain swapping in vitro. In order to understand the mechanism of selective dimerization among Rel subunits, we have determined the x-ray crystal structures of five RelB dimers. We find that RelB forms canonical side-by-side heterodimers with p50 and p52. We observe that, although mutation of four surface hydrophobic residues that are unique to RelB does not affect its propensity to form homodimers via domain swapping, alteration of two interfacial residues converts RelB to a side-by-side homodimer. Surprisingly, these mutant RelB homodimers remain distinct from canonical side-by-side NF-κB dimers in that the two monomers move away from one another along the 2-fold axis to avoid non-complementary interactions at the interface. The presence of distinct residues buried within the hydrophobic core of the RelB dimerization domain appears to influence the conformations of the surface residues that mediate the dimer interface. This conclusion is consistent with prior observations that alterations of domain core residues change dimerization propensity in the NF-κB family transcription factors. We suggest that RelB has evolved into a specialized NF-κB subunit with unique amino acids optimized for selective formation of heterodimers with p50 and p52.