Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies.

Some antibodies exhibit elevated viscosity at high concentrations, making them poorly suited for therapeutic applications requiring administration by injection such as subcutaneous or ocular delivery. Here we studied an anti-IL-13/IL-17 bispecific IgG4 antibody, which has anomalously high viscosity compared to its parent monospecific antibodies. The viscosity of the bispecific IgG4 in solution was decreased by only ~30% in the presence of NaCl, suggesting electrostatic interactions are insufficient to fully explain the drivers of viscosity. Intriguingly, addition of arginine-HCl reduced the viscosity of the bispecific IgG4 by ~50% to its parent IgG level. These data suggest that beyond electrostatics, additional types of interactions such as cation-π and/or π-π may contribute to high viscosity more significantly than previously understood. Molecular dynamics simulations of antibody fragments in the mixed solution of free arginine and explicit water were conducted to identify hotspots involved in self-interactions. Exposed surface aromatic amino acids displayed an increased number of contacts with arginine. Mutagenesis of the majority of aromatic residues pinpointed by molecular dynamics simulations effectively decreased the solution's viscosity when tested experimentally. This mutational method to reduce the viscosity of a bispecific antibody was extended to a monospecific anti-GCGR IgG1 antibody with elevated viscosity. In all cases, point mutants were readily identified that both reduced viscosity and retained antigen-binding affinity. These studies demonstrate a new approach to mitigate high viscosity of some antibodies by mutagenesis of surface-exposed aromatic residues on complementarity-determining regions that may facilitate some clinical applications.

Structure, function, and ion-binding properties of a K+ channel stabilized in the 2,4-ion-bound configuration.

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion-bound configuration (i.e., water, K+, water, K+-ion-bound configuration) of the K+ channel's selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type-like ion selectivity and apparent K+-binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K+ channel's selectivity filter, which fully agrees with the water-K+-ion-coupled transport detected by streaming potential measurements.

Inverted allosteric coupling between activation and inactivation gates in K+ channels.

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel's selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K+ channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K+-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel's selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel's selectivity filter.

CW-EPR Spectroscopy and Site-Directed Spin Labeling to Study the Structural Dynamics of Ion Channels.

Continuous-wave electron paramagnetic resonance spectroscopy (CW-EPR) and site-directed spin labeling (SDSL) are proven experimental approaches to assess the structural dynamics of proteins in general (Hubbell et al., Curr Opin Struct Biol 8(5):649-656, 1998; Kazmier et al., Curr Opin Struct Biol 45:100-108, 2016; Perozo et al., Science 285(5424):73-78, 1999). These techniques have been particularly effective assessing the structure of integral membrane proteins embedded in a lipid bilayer (Cortes et al., J Gen Physiol 117(2):165-180, 2001; Cuello et al., Science 306(5695):491-495, 2004; Dalmas et al., Structure 18(7):868-878, 2010; Li et al., Proc Natl Acad Sci U S A 112(44):E5926-5935, 2015; Perozo et al., J Gen Physiol 118(2):193-206, 2001), as well as determining the conformational changes associated with their biological function (Kazmier et al., Curr Opin Struct Biol 45:100-108, 2016; Perozo et al., Science 285(5424):73-78, 1999; Arrigoni et al., Cell 164(5):922-936, 2016; Dalmas et al., Nat Commun 5:3590, 2014; Dong et al., Science 308(5724):1023-1028, 2005; Farrens et al., Science 274(5288):768-770, 1996; Perozo et al., Nat Struct Biol 5(6):459-469, 1998; Perozo et al., Nature 418(6901):942-948, 2002). In this chapter, we described a practical guide for the spin-labeling, liposome reconstitution, and CW-EPR measurements of the prototypical bacterial K+ channel, KcsA.

Hysteresis of KcsA potassium channel's activation- deactivation gating is caused by structural changes at the channel's selectivity filter.

Mode-shift or hysteresis has been reported in ion channels. Voltage-shift for gating currents is well documented for voltage-gated cation channels (VGCC), and it is considered a voltage-sensing domain's (VSD) intrinsic property. However, uncoupling the Shaker K+ channel's pore domain (PD) from the VSD prevented the mode-shift of the gating currents. Consequently, it was proposed that an open-state stabilization of the PD imposes a mechanical load on the VSD, which causes its mode-shift. Furthermore, the mode-shift displayed by hyperpolarization-gated cation channels is likely caused by structural changes at the channel's PD similar to those underlying C-type inactivation. To demonstrate that the PD of VGCC undergoes hysteresis, it is imperative to study its gating process in the absence of the VSD. A back-door strategy is to use KcsA (a K+ channel from the bacteria Streptomyces lividans) as a surrogate because it lacks a VSD and exhibits an activation coupled to C-type inactivation. By directly measuring KcsA's activation gate opening and closing in conditions that promote or halt C-type inactivation, we have found (i) that KcsA undergoes mode-shift of gating when having K+ as the permeant ion; (ii) that Cs+ or Rb+, known to halt C-inactivation, prevented mode-shift of gating; and (iii) that, in the total absence of C-type inactivation, KcsA's mode-shift was prevented. Finally, our results demonstrate that an allosteric communication causes KcsA's activation gate to "remember" the conformation of the selectivity filter, and hence KcsA requires a different amount of energy for opening than for closing.

An improved method for the cost-effective expression and purification of large quantities of KcsA.

KcsA, the bacterial K(+) channel from Streptomyces lividans, is the prototypical model system to study the functional and structural correlations of the pore domain of eukaryotic voltage-gated K(+) channels (Kv channels). It contains all the molecular elements responsible for ion conduction, activation, deactivation and inactivation gating [1]. KcsA's structural simplicity makes it highly amenable for structural studies. Therefore, it is methodological advantageous to produce large amounts of functional and properly folded KcsA in a cost-effective manner. In the present study, we show an optimized protocol for the over-expression and purification of large amounts of high-quality, fully functional and crystallizable KcsA using inexpensive detergents, which significantly lowered the cost of the purification process.

Voltage-dependent BK and Hv1 channels expressed in non-excitable tissues: New therapeutics opportunities as targets in human diseases.

Voltage-gated ion channels are the molecular determinants of cellular excitability. This group of ion channels is one of the most important pharmacological targets in excitable tissues such as nervous system, cardiac and skeletal muscle. Moreover, voltage-gated ion channels are expressed in non-excitable cells, where they mediate key cellular functions through intracellular biochemical mechanisms rather than rapid electrical signaling. This review aims at illustrating the pharmacological impact of these ion channels, highlighting in particular the structural details and physiological functions of two of them - the high conductance voltage- and Ca(2+)-gated K(+) (BK) channels and voltage-gated proton (Hv1) channels- in non-excitable cells. BK channels have been implicated in a variety of physiological processes ranging from regulation of smooth muscle tone to modulation of hormone and neurotransmitter release. Interestingly, BK channels are also involved in modulating K(+) transport in the mammalian kidney and colon epithelium with a potential role in the hyperkalemic phenotype observed in patients with familial hyperkalemic hypertension type 2, and in the pathophysiology of hypertension. In addition, BK channels are responsible for resting and stimulated Ca(2+)-activated K(+) secretion in the distal colon. Hv1 channels have been detected in many cell types, including macrophages, blood cells, lung epithelia, skeletal muscle and microglia. These channels have a central role in the phagocytic system. In macrophages, Hv1 channels participate in the generation of reactive oxygen species in the respiratory burst during the process of phagocytosis.