Structural basis for enhanced HIV-1 neutralization by a dimeric immunoglobulin G form of the glycan-recognizing antibody 2G12.

The human immunoglobulin G (IgG) 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1) envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc) regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab)2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12.

A comitative source for object markers in Sinitic languages: kai55 in Waxiang and kang7 in Southern Min.

This analysis sets out to specifically discuss the polyfunctionality of [kai55] in Waxiang (Sinitic), whose lexical source is the verb 'to follow'. Amongst its various uses, we find a preposition 'with, along', a marker of adjuncts and a NP conjunction, thus superficially resembling its Mandarin cognate gen 'with'. Curiously, however, it has also evolved into a direct object marker in Waxiang, with a function similar to that of preposition ba < 'hold, take' as found in the S-ba-O-VP or so-called 'disposal' form in standard Mandarin. The pathways of grammaticalization for [kai55] in Waxiang are thus discussed in order to determine how it has developed this unusual grammatical function in one of the linguistic zones of China where verbs of giving or taking are, in fact, the main source for grammaticalized object markers in 'disposal' constructions. On the basis of 16th and 17th century Southern Min literature (Sinitic), a comparison is also made with analogous developments for comitative gòng 'with' to provide support for our hypothesis that the direct object marking use has evolved from the oblique function of a benefactive or dative, and is clearly separate from the crosslinguistically well-attested pathway that leads to its use as a conjunction. We would thus like to propose that these data contribute a new pattern to the stock of grammaticalization pathways, specifically, comitative > dative/benefactive > accusative (direct object marker).

Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the beta2a subunit of voltage-gated Ca2+ channels at Thr498 to facilitate cardiac L-type Ca2+ channels. CaMKII colocalizes with beta2a in cardiomyocytes and also binds to a domain in beta2a that contains Thr498 and exhibits an amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter, C. E. et al. (2006) Mol. Cell 23, 641). Here, we explore the selectivity of the actions of CaMKII among Ca2+ channel beta subunit isoforms. CaMKII phosphorylates the beta1b, beta2a, beta3, and beta4 isoforms with similar initial rates and final stoichiometries of 6-12 mol of phosphate per mol of protein. However, activated/autophosphorylated CaMKII binds to beta1b and beta2a with a similar apparent affinity but does not bind to beta3 or beta4. Prephosphorylation of beta1b and beta2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in beta2a are highly conserved in beta1b but are different in beta3 and beta4. Site-directed mutagenesis of this domain in beta2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-beta2a complexes in vitro and reduces interactions of CaMKII with beta2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with beta2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the Ca2+ channel beta subunits with CaMKII.