Karla K. Rodgers

Crystal structure of the RAG1 dimerization domain reveals multiple zinc-binding motifs including a novel zinc binuclear cluster.

The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together form a single structural domain. The domain also contains a unique zinc binuclear cluster in place of a normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helices that form the dimer interface.

Putting the pieces together: identification and characterization of structural domains in the V(D)J recombination protein RAG1.

Summary:  V(D)J recombination generates functional immunoglobulin and T‐cell receptor genes in developing lymphocytes. The recombination‐activating gene 1 (RAG1) and RAG2 proteins catalyze site‐specific DNA cleavage in this recombination process. Biochemical studies have identified catalytically active regions of each protein, referred to as the core regions. Here, we review our progress in the identification and characterization, in biophysical and biochemical terms, of topologically independent domains within both the non‐core and core regions of RAG1. Previous characterizations of a structural domain identified in the non‐core region of RAG1 from residues 265–380, referred to as the zinc‐binding dimerization domain, are discussed. This domain contains two zinc‐binding motifs, a RING finger and a C2H2 zinc finger. Core RAG1 also consists of multiple domains, each of which functions individually in one or more of the essential macromolecular interactions formed by the intact core protein. Two structural domains referred to as the central and the C‐terminal domains that include residues 528–760 and 761–979 of RAG1, respectively, have been identified. The interactions of the central and C‐terminal domains in core RAG1 with the recombination signal sequence (RSS) have contributed additional insight to a developing model for the RAG1–RSS complex.

The Central Domain of Core RAG1 Preferentially Recognizes Single-stranded Recombination Signal Sequence Heptamer

RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double strand breaks between each selected gene segment and its bordering recombination signal sequence (RSS) in a two-step mechanism in which the DNA is first nicked, followed by hairpin formation. The RSS consists of a conserved nonamer and heptamer sequence, in which the latter borders the site of DNA cleavage. A region within RAG1, referred to as the central domain (residues 528–760 of 1040 in the full-length protein), has been shown previously to bind specifically to the double-stranded (ds) RSS heptamer, but with both weak specificity and affinity. However, additional investigations into the RAG1-RSS heptamer interaction are required because the DNA substrate forms intermediate conformations during the V(D)J recombination reaction. These include the nicked and hairpin products, as well as likely base unpairing to produce single-stranded (ss) DNA near the cleavage site. Here, it was determined that although the central domain showed substantially higher binding affinity for ss and nicked versus ds substrate, the interaction with ss RSS was particularly robust. In addition, the central domain bound with greater sequence specificity to the ss RSS heptamer than to the ds form. This study provides important insight into the V(D)J recombination reaction, specifically that significant interaction of the RSS heptamer with RAG1 occurs only after the induction of conformational changes at the RSS heptamer.

A Non-Sequence-Specific DNA Binding Mode of RAG1 Is Inhibited by RAG2

RAG1 and RAG2 proteins catalyze site-specific DNA cleavage reactions in V(D)J recombination, a process that assembles antigen receptor genes from component gene segments during lymphocyte development. The first step towards the DNA cleavage reaction is the sequence-specific association of the RAG proteins with the conserved recombination signal sequence (RSS), which flanks each gene segment in the antigen receptor loci. Questions remain as to the contribution of each RAG protein to recognition of the RSS. For example, while RAG1 alone is capable of recognizing the conserved elements of the RSS, it is not clear if or how RAG2 may enhance sequence-specific associations with the RSS. To shed light on this issue, we examined the association of RAG1, with and without RAG2, with consensus RSS versus non-RSS substrates using fluorescence anisotropy and gel mobility shift assays. The results indicate that while RAG1 can recognize the RSS, the sequence-specific interaction under physiological conditions is masked by a high-affinity non-sequence-specific DNA binding mode. Significantly, addition of RAG2 effectively suppressed the association of RAG1 with non-sequence-specific DNA, resulting in a large differential in binding affinity for the RSS versus the non-RSS sites. We conclude that this represents a major means by which RAG2 contributes to the initial recognition of the RSS and that, therefore, association of RAG1 with RAG2 is required for effective interactions with the RSS in developing lymphocytes.

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin‐coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA–Rabaptin5 interaction is believed to function in the fusion of trans‐Golgi‐derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT‐binding domain. In this structure, the Rabaptin5 domain is a 90‐residue‐long helix. At the N‐terminal end, it forms a parallel coiled‐coil homodimer, which binds one GAT domain of GGA1. In the C‐terminal region, it further assembles into a four‐helix bundle tetramer. The Rabaptin5‐binding motif of the GGA1 GAT domain consists of a three‐helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle–helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four‐helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.

DNA Cleavage of a Cryptic Recombination Signal Sequence by RAG1 and RAG2 IMPLICATIONS FOR PARTIAL VH GENE REPLACEMENT

Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement. We have characterized the ability of the RAG proteins to bind and cleave a cryptic RSS (cRSS) located within an Ig VH gene segment. The RAG proteins bound with sequence specificity to either the consensus RSS or the cRSS. The RAG proteins nick the cRSS on both the top and bottom strands, thereby bypassing the formation of the DNA hairpin intermediate observed in RAG cleavage of canonical RSS substrates. We propose that the RAG proteins may utilize an alternative mechanism for double-stranded DNA cleavage, depending on the substrate sequence. These results have implications for further diversification of the antigen receptor repertoire as well as the role of the RAG proteins in genomic instability.

DNA Cleavage Activity of the V(D)J Recombination Protein RAG1 Is Autoregulated

ABSTRACT RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.

The disease-causing mutations in the carboxyl terminus of the cone cyclic nucleotide-gated channel CNGA3 subunit alter the local secondary structure and interfere with the channel active conformational change.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransducton. Mutations in the channel subunits are associated with achromatopsia and progressive cone dystrophy in humans. More than 50 mutations have been identified in the channel CNGA3 subunit, with 50% of them located in the carboxyl (C) terminus. This study investigates the defects of the two frequently occurring mutations, R377W and F488L, in the C-terminus of CNGA3. Ratiometric measurement of the intracellular Ca(2+) concentration and electrophysiological recordings showed the loss of functional activity of the mutant channels in an HEK293 heterologous expression system. Immunofluorescence labeling revealed an apparent cytosolic aggregation of the mutant channels compared to the wild type (WT). The R377W and F488L mutants, expressed and purified from Escherichia coli as glutathione S-transferase (GST) fused to the CNGA3 C-terminal domain, showed no negative effects on interactions with the channel subunits. Circular dichroism spectrum analyses were performed to examine the structural impact of the mutations. Although the R377W and F488L C-termini mutants retained stable, folded structures, the secondary structures of both mutants differed from the WT protein. Furthermore, the WT C-terminus exhibited a significant decrease in alpha-helical content in response to the channel ligands, while this allosteric transition was diminished in the two mutants. This is the first study showing the structural impact of the disease-causing mutations in the cone CNG channel subunit. The observed alterations in the local secondary structure and active conformational change may confer an adverse effect on the channels activity and cellular processing.

A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn(2+) ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn(2+) ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn(2+) ions. Stripping the full complement of bound Zn(2+) ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn(2+) core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn(2+) ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn(2+) ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn(2+) coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn(2+) serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.

Glycosylation of Skp1 affects its conformation and promotes binding to a model f-box protein.

In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased β-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells.