HMGB1 and RAGE in inflammation and cancer.

The immune system has evolved to respond not only to pathogens, but also to signals released from dying cells. Cell death through necrosis induces inflammation, whereas apoptotic cell death provides an important signal for tolerance induction. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein, released actively following cytokine stimulation as well as passively during cell death; it is the prototypic damage-associated molecular pattern (DAMP) molecule and has been implicated in several inflammatory disorders. HMGB1 can associate with other molecules, including TLR ligands and cytokines, and activates cells through the differential engagement of multiple surface receptors including TLR2, TLR4, and RAGE. RAGE is a multiligand receptor that binds structurally diverse molecules, including not only HMGB1, but also S100 family members and amyloid-beta. RAGE activation has been implicated in sterile inflammation as well as in cancer, diabetes, and Alzheimer's disease. While HMGB1 through interactions with TLRs may also be important, this review focuses on the role of the HMGB1-RAGE axis in inflammation and cancer.

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Mutual promotion of co-condensation of KRAS G-quadruplex and a well-folded protein HMGB1.

Liquid-liquid phase separation (LLPS) of G-quadruplex (GQ) is involved in many crucial cellular processes, while the quadruplex-folding and their functions are typically modulated by specific DNA-binding proteins. However, the regulatory mechanism of binding proteins, particularly the well-folded proteins, on the LLPS of GQs is largely unknown. Here, we investigated the effect of HMGB1 on the condensation of a G-quadruplex of KRAS promoter (GQKRAS). The results show that these two rigid macro-biomolecules undergo co-condensation through a mutual promotion manner, while neither of them can form LLPS alone. Fluidity measurements confirm that the liquid-like droplets are highly dynamic. HMGB1 facilitates and stabilizes the quadruplex folding of GQKRAS, and this process enhances their co-condensation. The KRAS promoter DNA retains quadruplex folding in the droplets; interference with the GQ-folding disrupts the co-condensation of GQKRAS/HMGB1. Mechanistic studies reveal that electrostatic interaction is a key driving force of the interaction and co-condensation of GQKRAS/HMGB1; meanwhile, the recognition of two macro-biomolecules plays a crucial role in this process. This result indicates that the phase separation of GQs can be modulated by DNA binding proteins, and this process could also be an efficient way to recruit specific DNA binding proteins.

Influence of an Intrinsically Disordered Region on Protein Domains Revealed by NMR-Based Electrostatic Potential Measurements.

Many human proteins possess intrinsically disordered regions containing consecutive aspartate or glutamate residues ("D/E repeats"). Approximately half of them are DNA/RNA-binding proteins. In this study, using nuclear magnetic resonance (NMR) spectroscopy, we investigated the electrostatic properties of D/E repeats and their influence on folded domains within the same protein. Local electrostatic potentials were directly measured for the HMGB1 protein, its isolated D/E repeats, and DNA-binding domains by NMR. The data provide quantitative information about the electrostatic interactions between distinct segments of HMGB1. Due to the interactions between the D/E repeats and the DNA-binding domains, local electrostatic potentials of the DNA-binding domains within the full-length HMGB1 protein were largely negative despite the presence of many positively charged residues. Our NMR data on counterions and electrostatic potentials show that the D/E repeats and DNA have similar electrostatic properties and compete for the DNA-binding domains. The competition promotes dissociation of the protein-DNA complex and influences the molecular behavior of the HMGB1 protein. These effects may be general among the DNA/RNA-binding proteins with D/E repeats.

Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA.

Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNA-binding capacity and forms lesions that lead to cancer cell death. However, the repair of these lesions compromises cisplatin's effectiveness. This study investigates how phosphorylation of HMGB1, a nuclear protein, modifies its binding to cisplatin-modified DNA (CP-DNA) and thus protects it from repair. Despite numerous methods for detecting protein-DNA interactions, quantitative approaches for understanding their molecular mechanism remain limited. Here, we applied click chemistry-based single-molecule force spectroscopy, achieving high-precision quantification of the interaction between phosphorylated HMGB1 and CP-DNA. This method utilizes a synergy of click chemistry and enzymatic ligation for precise DNA-protein immobilization and interaction in the system. Our results revealed that HMGB1 binds to CP-DNA with a significantly high rupture force of ∼130 pN, stronger than most natural DNA-protein interactions and varying across different DNA sequences. Moreover, Ser14 is identified as the key phosphorylation site, enhancing the interaction's kinetic stability by 35-fold. This increase in stability is attributed to additional hydrogen bonding suggested by molecular dynamics (MD) simulations. Our findings not only reveal the important role of phosphorylated HMGB1 in potentially improving cisplatin's therapeutic efficacy but also provide a precise method for quantifying protein-DNA interactions.

Investigation of N-Glycan Functions in Receptor for Advanced Glycation End Products V Domain through Chemical Glycoprotein Synthesis.

The receptor for advanced glycation end products (RAGE) plays a crucial role in inflammation-related pathways and various chronic diseases. Despite the recognized significance of N-glycosylation in the ligand-binding V domain (VD) of RAGE, a comprehensive understanding of the site-activity and structure-activity relationships is lacking due to the challenges in obtaining homogeneous glycoprotein samples through biological expression. Here, we combined chemical and chemoenzymatic approaches to synthesize RAGE-VD and its congeners with Asn3-glycosylation by incorporating precise N-glycan structures. Evaluation of these samples revealed that, in comparison to other RAGE-VD forms, α2,6-sialylated N-glycosylation at the Asn3 site results in more potent inhibition of HMGB1-induced nuclear factor-κB (NF-κB) expression in RAGE-overexpressing cells. Hydrogen/deuterium exchange-mass spectrum analysis revealed a sialylated RAGE-VD-induced interaction region within HMGB1. Conversely, Asn3 N-glycosylation in VD has negligible effects on RAGE-VD/S100B interactions. This study established an approach for accessing homogeneously glycosylated RAGE-VD and explored the modulatory effects of N-glycosylation on the interactions between RAGE-VD and its ligand proteins.

Structural basis for the activation and suppression of transposition during evolution of the RAG recombinase.

Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with methionine at residue 848 and cryo-electron microscopy, we determined structures that capture RAG engaged with transposon ends and U-shaped target DNA prior to integration (the target capture complex) and two forms of the RAG strand transfer complex that differ based on whether target site DNA is annealed or dynamic. Target site DNA base unstacking, flipping, and melting by RAG1 methionine 848 explain how this residue activates transposition, how RAG can stabilize sharp bends in target DNA, and why replacement of residue 848 by arginine during RAG domestication led to suppression of transposition activity. RAG2 extends a jawed vertebrate-specific loop to interact with target site DNA, and functional assays demonstrate that this loop represents another evolutionary adaptation acquired during RAG domestication to inhibit transposition. Our findings identify mechanistic principles of the final step in cut-and-paste transposition and the molecular and structural logic underlying the transformation of RAG from transposase to recombinase.

BAP1 forms a trimer with HMGB1 and HDAC1 that modulates gene × environment interaction with asbestos.

Carriers of heterozygous germline BAP1 mutations (BAP1+/-) are affected by the "BAP1 cancer syndrome." Although they can develop almost any cancer type, they are unusually susceptible to asbestos carcinogenesis and mesothelioma. Here we investigate why among all carcinogens, BAP1 mutations cooperate with asbestos. Asbestos carcinogenesis and mesothelioma have been linked to a chronic inflammatory process promoted by the extracellular release of the high-mobility group box 1 protein (HMGB1). We report that BAP1+/- cells secrete increased amounts of HMGB1, and that BAP1+/- carriers have detectable serum levels of acetylated HMGB1 that further increase when they develop mesothelioma. We linked these findings to our discovery that BAP1 forms a trimeric protein complex with HMGB1 and with histone deacetylase 1 (HDAC1) that modulates HMGB1 acetylation and its release. Reduced BAP1 levels caused increased ubiquitylation and degradation of HDAC1, leading to increased acetylation of HMGB1 and its active secretion that in turn promoted mesothelial cell transformation.

Structural Characteristics of High-Mobility Group Proteins HMGB1 and HMGB2 and Their Interaction with DNA.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.

High-mobility group box-1 and inter-alpha inhibitor proteins: In vitro binding and co-localization in cerebral cortex after hypoxic-ischemic injury.

The high-mobility group box-1 (HMGB1) protein is a transcription-regulating protein located in the nucleus. However, it serves as a damage-associated molecular pattern protein that activates immune cells and stimulates inflammatory cytokines to accentuate neuroinflammation after release from damaged cells. In contrast, Inter-alpha Inhibitor Proteins (IAIPs) are proteins with immunomodulatory effects including inhibition of pro-inflammatory cytokines. We have demonstrated that IAIPs exhibit neuroprotective properties in neonatal rats exposed to hypoxic-ischemic (HI) brain injury. In addition, previous studies have suggested that the light chain of IAIPs, bikunin, may exert its anti-inflammatory effects by inhibiting HMGB1 in a variety of different injury models in adult subjects. The objectives of the current study were to confirm whether HMGB1 is a target of IAIPs by investigating the potential binding characteristics of HMGB1 and IAIPs in vitro, and co-localization in vivo in cerebral cortices after exposure to HI injury. Solid-phase binding assays and surface plasmon resonance (SPR) were used to determine the physical binding characteristics between IAIPs and HMGB1. Cellular localizations of IAIPs-HMGB1 in neonatal rat cortex were visualized by double labeling with anti-IAIPs and anti-HMGB1 antibodies. Solid-phase binding and SPR demonstrated specific binding between IAIPs and HMGB1 in vitro. Cortical cytoplasmic and nuclear co-localization of IAIPs and HMGB1 were detected by immunofluorescent staining in control and rats immediately and 3 hours after HI. In conclusion, HMGB1 and IAIPs exhibit direct binding in vitro and co-localization in vivo in neonatal rats exposed to HI brain injury suggesting HMGB1 could be a target of IAIPs.

Paeonol attenuates inflammation by confining HMGB1 to the nucleus.

Inflammation is a biological process that exists in a large number of diseases. If the magnitude or duration of inflammation becomes uncontrolled, inflammation may cause pathological damage to the host. HMGB1 and NF-κB have been shown to play pivotal roles in inflammation-related diseases. New drugs aimed at inhibiting HMGB1 expression have become a key research focus. In the present study, we showed that paeonol (Pae), the main active component of Paeonia suffruticosa, decreases the expression of inflammatory cytokines and inhibits the translocation of HMGB1 induced by lipopolysaccharide (LPS). By constructing HMGB1-overexpressing (HMGB1+ ) and HMGB1-mutant (HMGB1m ) RAW264.7 cells, we found that the nuclear HMGB1 could induce an LPS-tolerant state in RAW264.7 cells and that paeonol had no influence on the expression of inflammatory cytokines in HMGB1m RAW264.7 cells. In addition, the anti-inflammatory property of paeonol was lost in HMGB1 conditional knockout mice, indicating that HMGB1 is a target of paeonol and a mediator through which paeonol exerts its anti-inflammatory function. Additionally, we also found that HMGB1 and P50 competitively bound with P65, thus inactivating the NF-κB pathway. Our research confirmed the anti-inflammation property of paeonol and suggests that inhibiting the translocation of HMGB1 could be a new strategy for treating inflammation.

Specific detection of high mobility group box 1 degradation product with a novel ELISA.

BACKGROUND: During sepsis or sterile tissue injury, the nuclear protein high mobility group box 1 (HMGB1) can be released to the extracellular space and ultimately into systemic circulation, where it mediates systemic inflammation and remote organ failure. The proinflammatory effects of HMGB1 can be suppressed by recombinant thrombomodulin (rTM), in part through a mechanism involving thrombin-rTM-mediated degradation of HMGB1. Given that HMGB1 is proinflammatory but the HMGB1 degradation product (desHMGB1) is not, an analytical method that discriminates between these two molecules may provide a more in-depth understanding of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency. METHODS: A peptide that has a shared amino-terminal structure with desHMGB1 was synthesized. C3H/lpr mice were immunized with the desHMGB1 peptide conjugate, and antibody-secreting hybridoma cells were developed using conventional methods. The reactivity and specificity of the antibodies were then analyzed using antigen-coated enzyme-linked immunosorbent assay (ELISA) as well as antibody-coated ELISA. Next, plasma desHMGB1 levels were examined in a cecal ligation and puncture (CLP)-induced septic mouse model treated with rTM. RESULTS: Through a series of screening steps, we obtained a monoclonal antibody that recognized desHMGB1 but did not recognize intact HMGB1. ELISA using this antibody specifically detected desHMGB1, which was significantly increased in CLP-induced septic mice treated with rTM compared with those treated with saline. CONCLUSIONS: In this study, we obtained a desHMGB1-specific monoclonal antibody. ELISA using the novel monoclonal antibody may be an option for the in-depth analysis of HMGB1-induced pathogenicity as well as rTM-mediated therapeutic efficiency.

2-O, 3-O desulfated heparin (ODSH) increases bacterial clearance and attenuates lung injury in cystic fibrosis by restoring HMGB1-compromised macrophage function.

BACKGROUND: High mobility group box 1 protein (HMGB1) is an alarmin following its release by immune cells upon cellular activation or stress. High levels of extracellular HMGB1 play a critical role in impairing the clearance of invading pulmonary pathogens and dying neutrophils in the injured lungs of cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS). A heparin derivative, 2-O, 3-O desulfated heparin (ODSH), has been shown to inhibit HMGB1 release from a macrophage cell line and is efficacious in increasing bacterial clearance in a mouse model of pneumonia. Thus, we hypothesized that ODSH can attenuate the bacterial burden and inflammatory lung injury in CF and we conducted experiments to determine the underlying mechanisms. METHODS: We determined the effects of ODSH on lung injury produced by Pseudomonas aeruginosa (PA) infection in CF mice with the transmembrane conductance regulator gene knockout (CFTR-/-). Mice were given ODSH or normal saline intraperitoneally, followed by the determination of the bacterial load and lung injury in the airways and lung tissues. ODSH binding to HMGB1 was determined using surface plasmon resonance and in silico docking analysis of the interaction of the pentasaccharide form of ODSH with HMGB1. RESULTS: CF mice given 25 mg/kg i.p. of ODSH had significantly lower PA-induced lung injury compared to mice given vehicle alone. The CF mice infected with PA had decreased levels of nitric oxide (NO), increased levels of airway HMGB1 and HMGB1-impaired macrophage phagocytic function. ODSH partially attenuated the PA-induced alteration in the levels of NO and airway HMGB1 in CF mice. In addition, ODSH reversed HMGB1-impaired macrophage phagocytic function. These effects of ODSH subsequently decreased the bacterial burden in the CF lungs. In a surface plasmon resonance assay, ODSH interacted with HMGB1 with high affinity (KD = 3.89 × 10-8 M) and induced conformational changes that may decrease HMGB1's binding to its membrane receptors, thus attenuating HMGB1-induced macrophage dysfunction. CONCLUSIONS: The results suggest that ODSH can significantly decrease bacterial infection-induced lung injury in CF mice by decreasing both HMGB1-mediated impairment of macrophage function and the interaction of HMGB1 with membrane receptors. Thus, ODSH could represent a novel approach for treating CF and ARDS patients that have HMGB1-mediated lung injury.

Insights into telomeric G-quadruplex DNA recognition by HMGB1 protein.

HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.

Proteins Binding to the Carbohydrate HNK-1: Common Origins?

The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1's importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.

In Situ Visualization of Proteins in Single Cells by Time-of-Flight-Secondary Ion Mass Spectrometry Coupled with Genetically Encoded Chemical Tags.

In situ visualization of proteins of interest in single cells is attractive in cell biology, molecular biology, and biomedicine fields. Time-of-flight-secondary ion mass spectrometry (ToF-SIMS) is a powerful tool for imaging small organic molecules in single cells, yet difficult to image biomacromolecules such as proteins and DNA. Herein, a universal strategy is reported to image specific proteins in single cells by ToF-SIMS following genetic incorporation of fluorine-containing unnatural amino acids as a chemical tag into the proteins via a genetic code expansion technique. The method was developed and validated by imaging a green fluorescence protein (GFP) in Escherichia coli (E. coli) and human HeLa cancer cells and then utilized to visualize the characteristic polar distribution of chemotaxis protein CheA in E. coli cells and the interaction between high-mobility group box 1 protein and cisplatin-damaged DNA in HeLa cells. The present work highlights the power of ToF-SIMS imaging combined with genetically encoded chemical tags for in situ visualization of specific proteins as well as the interactions between proteins and drugs or drug-damaged DNA in single cells.

High-mobility group box 1 protein (HMGB1) from Cherry Valley duck mediates signaling pathways and antiviral activity.

High-mobility group box 1 protein (HMGB1) shows endogenous damage-associated molecular patterns (DAMPs) and is also an early warning protein that activates the body's innate immune system. Here, the full-length coding sequence of HMGB1 was cloned from the spleen of Cherry Valley duck and analyzed. We find that duck HMGB1(duHMGB1) is mostly located in the nucleus of duck embryo fibroblast (DEF) cells under normal conditions but released into the cytoplasm after lipopolysaccharide (LPS) stimulation. Knocking-down or overexpressing duHMGB1 had no effect on the baseline apoptosis rate of DEF cells. However, overexpression increased weakly apoptosis after LPS activation. In addition, overexpression strongly activated the IFN-I/IRF7 signaling pathway in DEF cells and significantly increased the transcriptional level of numerous pattern recognition receptors (PRRs), pro-inflammatory cytokines (IL-6, TNF-α), IFNs and antiviral molecules (OAS, PKR, Mx) starting from 48 h post-transfection. Overexpression of duHMGB1 strongly impacted duck virus replication, either by inhibiting it from the first stage of infection for novel duck reovirus (NDRV) and at late stage for duck Tembusu virus (DTMUV) or duck plague virus (DPV), or promoting replication at early stage for DTMUV and DPV infection. Importantly, data from duHMGB1 overexpression and knockdown experiments, time-dependent DEF cells transcriptional immune responses suggest that duHMGB1 and RIG-I receptor might cooperate to promote the expression of antiviral proteins after NDRV infection, as a potential mechanism of duHMGB1-mediated antiviral activity.

Effect of in vivo post-translational modifications of the HMGB1 protein upon binding to platinated DNA: a molecular simulation study.

Cisplatin is one of the most widely used anticancer drugs. Its efficiency is unfortunately severely hampered by resistance. The High Mobility Group Box (HMGB) proteins may sensitize tumor cells to cisplatin by specifically binding to platinated DNA (PtDNA) lesions. In vivo, the HMGB/PtDNA binding is regulated by multisite post-translational modifications (PTMs). The impact of PTMs on the HMGB/PtDNA complex at atomistic level is here investigated by enhanced sampling molecular simulations. The PTMs turn out to affect the structure of the complex, the mobility of several regions (including the platinated site), and the nature of the protein/PtDNA non-covalent interactions. Overall, the multisite PTMs increase significantly the apparent synchrony of all the contacts between the protein and PtDNA. Consequently, the hydrophobic anchoring of the side chain of F37 between the two cross-linked guanines at the platinated site-a key element of the complexes formation - is more stable than in the complex without PTM. These differences can account for the experimentally measured greater affinity for PtDNA of the protein isoforms with PTMs. The collective behavior of multisite PTMs, as revealed here by the synchrony of contacts, may have a general significance for the modulation of intermolecular recognitions occurring in vivo.

Real-time analysis of RAG complex activity in V(D)J recombination.

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

[Identification of Distinct Amino Acid Composition of Human Cruciform Binding Proteins].

Cruciform structures are preferential targets for many architectural and regulatory proteins, as well as a number of DNA binding proteins with weak sequence specificity. Some of these proteins are also capable of inducing the formation of cruciform structures upon DNA binding. In this paper we analyzed the amino acid composition of eighteen cruciform binding proteins of Homo sapiens. Comparison with general amino acid frequencies in all human proteins revealed unique differences, with notable enrichment for lysine and serine and/or depletion for alanine, glycine, glutamine, arginine, tyrosine and tryptophan residues. Based on bootstrap resampling and fuzzy cluster analysis, multiple molecular mechanisms of interaction with cruciform DNA structures could be suggested, including those involved in DNA repair, transcription and chromatin regulation. The proteins DEK, HMGB1 and TOP1 in particular formed a very distinctive group. Nonetheless, a strong interaction network connecting nearly all the cruciform binding proteins studied was demonstrated. Data reported here will be very useful for future prediction of new cruciform binding proteins or even construction of predictive tool/web-based application.