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Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called “stereotypic RFs,” are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2–CH3 elbow in the canonical antigen-binding manner involving a large antigen–antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu(432)–His(435) region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

The archaeal exo-β-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal structure of GlmA in complex with glucosamine at 1.27 A resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 β-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 β-galactosidases. We found that upon dimerization, the active site of GlmA changes shape, enhancing its ability to hydrolyze the smaller substrate in a manner similar to that of homotrimeric GH42 β-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the “evolutionary heritage residue” it shares with GH35 β-galactosidase. Our study suggests that GH35 and GH42 β-galactosidases evolved by exploiting the structural features of GlmA.

Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriC-DnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.

Tapes japonica lysozyme (TJL) is classified as a member of the recently established i-type lysozyme family. In this study, we solved the crystal structure of TJL complexed with a trimer of N-acetylglucosamine to 1.6A resolution. Based on structure and mutation analyses, we demonstrated that Glu-18 and Asp-30 are the catalytic residues of TJL. Furthermore, the present findings suggest that the catalytic mechanism of TJL is a retaining mechanism that proceeds through a covalent sugar-enzyme intermediate. On the other hand, the quaternary structure in the crystal revealed a dimer formed by the electrostatic interactions of catalytic residues (Glu-18 and Asp-30) in one molecule with the positive residues at the C terminus in helix 6 of the other molecule. Gel chromatography analysis revealed that the TJL dimer remained intact under low salt conditions but that it dissociated to TJL monomers under high salt conditions. With increasing salt concentrations, the chitinase activity of TJL dramatically increased. Therefore, this study provides novel evidence that the lysozyme activity of TJL is modulated by its quaternary structure.

DnaA forms a homomultimeric complex with the origin of chromosomal replication (oriC) to unwind duplex DNA. The interaction of the DnaA N terminus with the DnaB helicase is crucial for the loading of DnaB onto the unwound region. Here, we determined the DnaA N terminus structure using NMR. This region (residues 1–108) consists of a rigid region (domain I) and a flexible region (domain II). Domain I has an α-α-β-β-α-β motif, similar to that of the K homology (KH) domain, and has weak affinity for oriC single-stranded DNA, consistent with KH domain function. A hydrophobic surface carrying Trp-6 most likely forms the interface for domain I dimerization. Glu-21 is located on the opposite surface of domain I from the Trp-6 site and is crucial for DnaB helicase loading. These findings suggest a model for DnaA homomultimer formation and DnaB helicase loading on oriC.

The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.