Despite the therapeutic success of monoclonal antibodies (mAbs), early identification of developable mAb-drug candidates with optimal manufacturability, stability, and delivery attributes remains elusive. Poor solution behavior, which manifests as high solution viscosity or opalescence, profoundly affects the developability of mAb-drugs. Employing a diverse dataset of 59 mAbs, including 43 approved products, and an array of molecular descriptors spanning colloidal, conformational, charge-based, hydrodynamic, and hydrophobic properties, we show that poor solution behavior is prevalent (>30%) in mAbs and is singularly predicted (>90%) by the diffusion interaction parameter (kD), a dilute-solution measure of colloidal self-interaction. No other descriptor, individually or in combination, was found to be as effective as kD. We also show that well-behaved mAbs, a significant subset of which bear high positive charge and pI, present no disadvantages with respect to pharmacokinetics in humans. Here, we provide a systematic framework with quantitative thresholds for selecting well-behaved therapeutic mAbs during drug-discovery.

The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear.Here we report an unexpected circular arrangement (ring) of SYT's cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18-43 nm, corresponding to 11-26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded. S ynaptotagmin-1 (SYT) is the calcium (Ca 2+ ) sensor that triggers synchronous release of neurotransmitters for synaptic transmission (1-4). It is a transmembrane protein, localized to the synaptic vesicles (1, 5), with tandem cytosolic C2 domains (C2A and C2B) that bind phospholipids in both a Ca 2+ -independent and a Ca 2+ -dependent manner (1, 6). The membrane-distal C2B domain interacts with acidic lipids such as phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to mediate efficient docking of the synaptic vesicles (7-10) before the influx of Ca 2+ ions. Recent studies (7,8,11,12) have located this calciumindependent membrane-binding site to a polybasic patch on the C2B domain (site I in Fig. 1A). The binding of Ca 2+ to the calcium-coordination pocket of the C2B domain (site II in Fig. 1A) triggers the insertion of flanking portions of site II into the plasma membrane, and this Ca 2+ -triggered membrane insertion is absolutely required for neurotransmitter release (13-16).The C2B domain also mediates the Ca 2+ -independent binding of SYT to neuronal soluble N-ethylmaleimide-sensitive factor activating protein receptor on the plasma membrane (t-SNARE) [syntaxin/ synaptosomal-associated protein 25 (SNAP25)], most likely via its interaction with SNAP25 (17-21). This interaction is believed to position SYT on the prefusion SNARE complexes to trigger rapid exocytosis in response to Ca 2+ (20,22). How the insertion of site II into the membrane bilayer is coupled to the completion of the SNARE assembly to release neurotransmitter is still unclear, although some key points have been established. In the prefusion state, the SNARE complexes...

Hormones and neurotransmitters are released through fluctuating exocytotic fusion pores that can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v-SNAREs and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not understood. Here, we probed the dilation of single fusion pores using v-SNARE-reconstituted ~23-nm-diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, 'flipped' t-SNAREs. Pore nucleation required a minimum of two v-SNAREs per NLP face, and further increases in v-SNARE copy numbers did not affect nucleation rate. By contrast, the probability of pore dilation increased with increasing v-SNARE copies and was far from saturating at 15 v-SNARE copies per face, the NLP capacity. Our experimental and computational results suggest that SNARE availability may be pivotal in determining whether neurotransmitters or hormones are released through a transient ('kiss and run') or an irreversibly dilating pore (full fusion).DOI: http://dx.doi.org/10.7554/eLife.22964.001

Signal peptidase I (SPase I) is critical for the release of translocated preproteins from the membrane as they are transported from a cytoplasmic site of synthesis to extracytoplasmic locations. These proteins are synthesized with an amino-terminal extension, the signal sequence, which directs the preprotein to the Sec-or Tat-translocation pathway. Recent evidence indicates that the SPase I cleaves preproteins as they emerge from either pathway, though the steps involved are unclear. Now that the structure of many translocation pathway components has been elucidated, it is critical to determine how these components work in concert to support protein translocation and cleavage. Molecular modeling and NMR studies have provided insight on how the preprotein docks on SPase I in preparation for cleavage. This is a key area for future work since SPase I enzymes in a variety of species have now been identified and the inhibition of these enzymes by antibiotics is being pursued. The eubacterial SPase I is essential for cell viability and belongs to a unique group of serine endoproteases which utilize a Ser-Lys catalytic dyad instead of the prototypical Ser-His-Asp triad used by eukaryotes. As such, SPase I is a desirable antimicrobial target. Advances in our understanding of how the preprotein interfaces with SPase I during the final stages of translocation will facilitate future development of inhibitors that display a high efficacy against SPase I function.

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (Otof Ala515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

The initial, nanometer-sized connection between the plasma membrane and a hormone- or neurotransmitter-filled vesicle –the fusion pore– can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics determine release and vesicle recycling kinetics, but pore properties are poorly known because biochemically defined single-pore assays are lacking. We isolated single flickering pores connecting v-SNARE-reconstituted nanodiscs to cells ectopically expressing cognate, “flipped” t-SNAREs. Conductance through single, voltage-clamped fusion pores directly reported sub-millisecond pore dynamics. Pore currents fluctuated, transiently returned to baseline multiple times, and disappeared ~6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, TMD modifications designed to disrupt v- and t-SNARE TMD zippering prolonged pore lifetimes dramatically. We propose that the post-fusion geometry of the proteins contribute to pore stability.

Identification of the signal peptide-binding domain within SecA ATPase is an important goal for understanding the molecular basis of SecA preprotein recognition as well as elucidating the chemomechanical cycle of this nanomotor during protein translocation. In this study, Förster resonance energy transfer methodology was employed to map the location of the SecA signal peptide-binding domain using a collection of functional monocysteine SecA mutants and alkaline phosphatase signal peptides labeled with appropriate donor-acceptor fluorophores. Fluorescence anisotropy measurements yielded an equilibrium binding constant of 1.4 or 10.7 μM for the alkaline phosphatase signal peptide labeled at residue 22 or 2, respectively, with SecA, and a binding stoichiometry of one signal peptide bound per SecA monomer. Binding affinity measurements performed with a monomerbiased mutant indicate that the signal peptide binds equally well to SecA monomer or dimer. Distance measurements determined for 13 SecA mutants show that the SecA signal peptide-binding domain encompasses a portion of the preprotein cross-linking domain but also includes regions of nucleotidebinding domain 1 and particularly the helical scaffold domain. The identified region lies at a multidomain interface within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel. Our FRET-mapped binding domain, in contrast to the domain identified by NMR spectroscopy, includes the two-helix finger that has been shown to interact with the preprotein during translocation and lies at the entrance to the protein-conducting channel in the recently determined SecA-SecYEG structure.Proteins are secreted across or integrated into biological membranes by means of a variety of protein translocation systems that have been characterized over the past several decades. In Escherichia coli, the major pathway for protein secretion is the general secretion (Sec) pathway that is composed of two fundamental components: the SecYEG heterotrimeric complex that comprises the protein-conducting channel and the SecA ATPase nanomotor that drives † This work was supported by Grants GM42033 and GM37639 from Figure S1), binding affinity of unlabeled SP2 and IANBD-labeled SP2 with IAEDANS-labeled SecA-Cys-256 ( Figure S2) and mapping of the FRET-determined signal peptide-binding site on the SecA crystal structures of B. subtilis, E. coli, T. thermophilus, and Mycobacterium tuberculosis ( Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 7. Recently, a model for preprotein translocation has been proposed on the basis of a SecASecYEG cocrystal structure (10) and disulfide cross-linking studies (11). In this model, SecA captures the preprotein in a clamp formed by nucleotide-binding domain 2 (NBD-2) 1 , the preprotein binding domain (PPXD), and the h...

Here we introduce ApoE-based Nanolipoprotein particles (NLP) – soluble, discoidal bilayer mimetic of ~23 nm in diameter, as fusion partners to study the dynamics of fusion pores induced by SNARE proteins. Using in vitro lipid mixing and content release assays, we report that NLPs reconstituted with synaptic v-SNARE VAMP2 (vNLP) fuse with liposomes containing the cognate t-SNARE (Syntaxin1/SNAP25) partner, with the resulting fusion pore opening directly to the external buffer. Efflux of encapsulated fluorescent dextrans of different size shows that unlike the smaller nanodiscs, these larger NLPs accommodate the expansion of the fusion pore to at least ~ 9 nm and dithionite quenching of fluorescent lipid introduced in vNLP confirms that the NLP fusion pores are short-lived and eventually reseal. The NLPs also have capacity to accommodate larger number of proteins and using vNLPs were defined number of VAMP2 protein, including physiologically relevant copy numbers, we find that 3–4 copies of VAMP2 (minimum 2 per face) are required to keep a nascent fusion pore open and the SNARE proteins act cooperatively to dilate the nascent fusion pore.