Chwan-Deng Hsiao

Background: Hepatitis B virus (HBV) infection persists through immune evasion strategies, including engagement of the inhibitory receptor CD33 (Siglec-3) by α2,6-linked sialoglycans on HBsAg. This interaction induces ITIM phosphorylation and SHP-1/2 recruitment, dampening myeloid cell activation. The monoclonal antibody 10C8 has been identified as a potent antagonist of CD33, but the structural basis for its inhibitory activity remains unclear. Methods: We determined the 3.2 Å crystal structure of the human CD33 extracellular domain (CD33-ECD) in complex with the Fab fragment of 10C8 (Fab-10C8). The stoichiometry and assembly of the complex in solution were validated by size-exclusion chromatography and analytical ultracentrifugation (AUC). Structural comparisons with apo CD33 were performed to assess potential Fab-induced conformational changes, and buried surface area analyses were conducted to characterize binding interfaces. Results: The structure reveals a 2:2 stoichiometry complex comprising two CD33-ECD molecules and two Fab-10C8 fragments, consistent with the 157 kDa molecular mass determined by AUC. Each Fab engages the V-set domain through extensive complementarity-determining region (CDR) interactions, burying ~612 Å ² of surface area, while inter-Fab contacts stabilize a compact dimeric arrangement of CD33-ECD. This geometry differs markedly from the relaxed apo state, showing a ~21° reduction in the dimer angle. The antibody-binding epitope is adjacent to, but not overlapping with, the canonical sialic acid-binding cleft, leading to steric occlusion that prevents HBsAg engagement. Together with a potential Fab-induced ectodomain compaction, this conformation restricts CD33 clustering and likely precludes SHP-1/2 recruitment, providing a mechanistic explanation for the antagonistic activity of 10C8. Conclusions: This study provides the structural insight into antibody-mediated inhibition of CD33. By locking CD33 into a sterically occluded, signaling-refractory conformation, 10C8 effectively reverses HBV-induced immunosuppression, thereby restoring host antiviral activity. These findings establish a structural framework for rational design of Siglec-targeted immunotherapies against chronic viral infections and other immune regulatory disorders.

CC2D1A is a multidomain scaffold protein implicated in transcriptional regulation and autosomal recessive non-syndromic intellectual disability (NSID), yet its molecular mechanism is still poorly understood due to a lack of structural information. Here, we present the crystal structure of the human CC2D1A491–810 fragment, encompassing the fourth DM14 domain, a coiled-coil region, and a C-terminal C2 domain. These elements form a compact, integrated architecture, with the C2 domain mediating symmetric dimerization through conserved electrostatic interactions. In addition, a unique antiparallel β1–β10 sheet connects the coiled-coil and C2 domains, stabilizing the tertiary structure. Fluorescence polarization assays reveal micromolar DNA-binding affinity, likely mediated by the basic surface of the DM14 domain. Comparison with the Drosophila homolog Lgd highlights conserved topology with added structural features, offering insights into CC2D1A’s vertebrate-specific functions and NSID-related mutations.

The nitrate transporter At NRT1.1 in Arabidopsis thaliana exhibits a unique dual‐affinity transport system that optimizes nitrate uptake across variable environmental conditions. Using molecular dynamics simulations, we investigate the dynamic mechanisms governing nitrate retention and transport of At NRT1.1. Our results reveal that protonated state of His356 stabilizes nitrate binding through interactions with Arg45, while deprotonation of His356 facilitates nitrate release toward the cytosol. We also observed that the loop between transmembrane helices TM4 and TM5, and the conserved FXXFYXXINXG motif in TM5 involved in conformational changes between inward‐open and occluded states. Specifically, the Glycine in this motif serves as a pivot point, enabling TM5 bending and promoting the formation of a hydrophobic layer between TM5 and TM10, thereby sealing the intracellular gate. Additionally, phosphorylation of Thr101 further modulates At NRT1.1 dynamic, enhancing flexibility in the TM2 and TM4 region and potentially influencing the probability of occluded state formation. These findings provide mechanistic insights into the sophisticated dual‐affinity transport system of At NRT1.1.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5–15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

The ParABS system, composed of ParA (an ATPase), ParB (a DNA binding protein), and parS (a centromere-like DNA), regulates bacterial chromosome partition. The ParB-parS partition complex interacts with the nucleoid-bound ParA to form the nucleoid-adaptor complex (NAC). In Helicobacter pylori, ParA and ParB homologs are encoded as HpSoj and HpSpo0J (HpParA and HpParB), respectively. We determined the crystal structures of the ATP hydrolysis deficient mutant, HpParAD41A, and the HpParAD41A-DNA complex. We assayed the CTPase activity of HpParB and identified two potential DNA binding modes of HpParB regulated by CTP, one is the specific DNA binding by the DNA binding domain and the other is the non-specific DNA binding through the C-terminal domain under the regulation of CTP. We observed an interaction between HpParAD41A and the N-terminus fragment of HpParB (residue 1–10, HpParBN10) and determined the crystal structure of the ternary complex, HpParAD41A-DNA-HpParBN10 complex which mimics the NAC formation. HpParBN10 binds near the HpParAD41A dimer interface and is clamped by flexible loops, L23 and L34, through a specific cation-π interaction between Arg9 of HpParBN10 and Phe52 of HpParAD41A. We propose a molecular mechanism model of the ParABS system providing insight into chromosome partition in bacteria.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA–DNA and SegB–DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon–helix–helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA–SegB–DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

Chronic hepatitis B (CHB) infection is rarely eradicated by current antiviral nucleos(t)ide analogues. We found that α2,6-biantennary sialoglycans of HBV surface antigen (HBsAg) bound human SIGLEC-3 (CD33) by IP and ELISA, and the binding affinity between SIGLEC-3 and α2,6-biantennary sialoglycans was determined by biolayer interferometry (equilibrium dissociation constant [KD]: 1.95 × 10^–10 ± 0.21 × 10^–10 M). Moreover, HBV activated SIGLEC-3 on myeloid cells and induced immunosuppression by stimulating immunoreceptor tyrosine-based inhibitory motif phosphorylation and SHP-1/-2 recruitment via α2,6-biantennary sialoglycans on HBsAg. An antagonistic anti–SIGLEC-3 mAb reversed this effect and enhanced cytokine production in response to TLR-7 agonist GS-9620 in PBMCs from CHB patients. Moreover, anti–SIGLEC-3 mAb alone was able to upregulate the expression of molecules involved in antigen presentation, such as CD80, CD86, CD40, MHC-I, MHC-II, and PD-L1 in CD14⁺ cells. Furthermore, SIGLEC-3 SNP rs12459419 C, which expressed a higher amount of SIGLEC-3, was associated with increased risk of hepatocellular carcinoma (HCC) in CHB patients (HR: 1.256, 95% CI: 1.027–1.535, P = 0.0266). Thus, blockade of SIGLEC-3 is a promising strategy to reactivate host immunity to HBV and lower the incidence of HCC in the CHB patient population.

DNA damages lead to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal protein PriA specifically recognizes DNA fork structure and recruits other primosomal proteins to load the replicative helicase, in order to re-establish the replication fork. PriA binding on DNA is the first step to restart replication forks for proper DNA repair. Using a single-molecule fluorescence colocalization experiment, we measured the thermodynamic and real-time kinetic properties of fluorescence-labeled Gram-positive bacteria Geobacillus stearothermophilus PriA binding on DNA forks. We showed that PriA preferentially binds to a DNA fork structure with a fully duplex leading strand at sub-nanomolar affinity (K_d = (268±99) pM). PriA binds dynamically, and its association and dissociation rate constants can be determined using the appearance and disappearance of the fluorescence signal. In addition, we showed that PriA binds to DNA forks as monomer using photobleaching step counting. This information offers a molecular basis essential for understanding the mechanism of replication restart.

DNA replication forks often encounter template DNA lesions that can stall their progression. The PriA-dependent pathway is the major replication restart mechanism in Gram-positive bacteria, and it requires several primosome proteins. Among them, PriA protein—a 3′ to 5′ superfamily-2 DNA helicase—is the key factor in recognizing DNA lesions and it also recruits other proteins. Here, we investigated the ATPase and helicase activities of Streptococcus pneumoniae PriA (SpPriA) through biochemical and kinetic analyses. By comparing various DNA substrates, we observed that SpPriA is unable to unwind duplex DNA with high GC content. We constructed a deletion mutant protein (SpPriA_deloop) from which the loop area of the DNA-binding domain of PriA had been removed. Functional assays on SpPriA_deloop revealed that the loop area is important in endowing DNA-binding properties on the helicase. We also show that presence of DnaD loader protein is important for enhancing SpPriA ATPase and DNA unwinding activities.