Carboxyl-terminal sequences in APOA5 are important for suppressing ANGPTL3/8 activity.

Apolipoprotein AV (APOA5) lowers plasma triglyceride (TG) levels by binding to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppressing its capacity to inhibit lipoprotein lipase (LPL) catalytic activity and its ability to detach LPL from binding sites within capillaries. However, the sequences in APOA5 that are required for suppressing ANGPTL3/8 activity have never been defined. A clue to the identity of those sequences was the presence of severe hypertriglyceridemia in two patients harboring an APOA5 mutation that truncates APOA5 by 35 residues ("APOA5Δ35"). We found that wild-type (WT) human APOA5, but not APOA5Δ35, suppressed ANGPTL3/8's ability to inhibit LPL catalytic activity. To pursue that finding, we prepared a mutant mouse APOA5 protein lacking 40 C-terminal amino acids ("APOA5Δ40"). Mouse WT-APOA5, but not APOA5Δ40, suppressed ANGPTL3/8's capacity to inhibit LPL catalytic activity and sharply reduced plasma TG levels in mice. WT-APOA5, but not APOA5Δ40, increased intracapillary LPL levels and reduced plasma TG levels in Apoa5-/- mice (where TG levels are high and intravascular LPL levels are low). Also, WT-APOA5, but not APOA5Δ40, blocked the ability of ANGPTL3/8 to detach LPL from cultured cells. Finally, an antibody against a synthetic peptide corresponding to the last 26 amino acids of mouse APOA5 reduced intracapillary LPL levels and increased plasma TG levels in WT mice. We conclude that C-terminal sequences in APOA5 are crucial for suppressing ANGPTL3/8 activity in vitro and for regulating intracapillary LPL levels and plasma TG levels in vivo.

Hydrogen-Deuterium Exchange Epitope Mapping of Glycosylated Epitopes Enabled by Online Immobilized Glycosidase.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is widely used for monoclonal antibody (mAb) epitope mapping, which aids in the development of therapeutic mAbs and vaccines, as well as enables the understanding of viral immune evasion. Numerous mAbs are known to recognize N-glycosylated epitopes and to bind in close proximity to an N-glycan site; however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on a solid resin and incorporated it into an online HDX-MS workflow for post-HDX deglycosylation. The resin-immobilized PNGase Dj exhibited robust tolerance to various buffer conditions and was employed in a column format that can be readily adapted into a typical HDX-MS platform. Using this system, we were able to obtain full sequence coverage of the SARS-CoV-2 receptor-binding domain (RBD) and map the glycosylated epitope of the glycan-binding mAb S309 to the RBD.

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif.

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics.

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cß) that increase the unfolding temperature of Cα/Cß by 20 °C, improve the expression of four separate α/ß TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cß mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.

Modulation of the Biophysical Properties of Bifunctional Antibodies as a Strategy for Mitigating Poor Pharmacokinetics.

Interest in the development of bi- or multispecific antibody (BsAbs)-based biotherapeutics is growing rapidly due to their inherent ability to interact with many targets simultaneously, thereby potentially protracting their functionality relative to monoclonal antibodies (mAbs). Biophysical property assays have been used to improve the probability of clinical success for various mAb therapeutics; however, there is a paucity of such data for BsAbs. This work evaluates a fusion of an IgG with an isolated protein domain (deemed ECD) and serves to understand how molecular architecture influences biophysical and biochemical properties and, in turn, how these relate to drug disposition. The biophysical characteristics of the molecules (charge, nonspecific binding, FcRn and Fcγ receptor interactions, thermal stability, structure-dynamics, and hydrophobic properties) indicated preferred orientations of ECD and IgG, which supported better pharmacokinetic outcomes. In certain instances, in which ECD-IgG configurations led to suboptimal biophysical behavior in the form of increased hydrophobicity and global ECD instability, drug clearance was found to be increased by ≥2-fold, driven by endothelial cell-based association/clearance mechanisms in the liver, kidneys, and spleen. Improvements in the pharmacokinetic properties were afforded by positional modulation of ECD that was able to bring the disposition characteristics in line with those of the parental mAb. The findings provide some pragmatic, broadly applicable strategies and guidance for the design considerations and evaluation of ECD-BsAb constructs. Additional studies, delineating the precise interactions involved in the clearance of the ECD-BsAb constructs, remain an opportunistic area for improving their in vivo kinetic properties.

The folding unit of phosphofructokinase-2 as defined by the biophysical properties of a monomeric mutant.

Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened ß-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 µM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240-309. These residues correspond to most of the ß-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the ß-meander module is not affected by monomerizing mutations.

How the ankyrin and SOCS box protein, ASB9, binds to creatine kinase.

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. The ASB proteins function as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that specifically transfer ubiquitin to cellular proteins targeting them for degradation by the proteasome. ASB9 binds to creatine kinase (CK) and targets it for degradation; however, the way in which ASB9 interacts with CK is not yet known. We present a complete characterization of the binding of ASB9 to CK. One ASB9 molecule binds to a dimer of CK. The binding affinity of ASB9(1-252) was extremely tight, and no dissociation could be observed. Deletion of the 34 N-terminal amino acids forming ASB9(35-252) resulted in weakening of the binding, so that a binding affinity of 2.6 nM could be measured. Amide hydrogen-deuterium exchange (HDXMS) experiments showed that both ASB9(1-252) and ASB9(35-252) protected the same region of CK, residues 182-203, which forms one side of the active site. The HDXMS experiments indicated that the N-terminal disordered region and first ankyrin repeat of ASB9 are protected from exchange in the complex. Molecular docking yielded a structural model consistent with all of the data that suggested the N-terminal residues of ASB9(1-252) may lie in one CK active site. This model was corroborated by enzymatic activity assays and mutational analysis.

The phylogenetics of dynamics in DNA clamp proteins.

Using HXMS to measure protein dynamics, Fang and colleagues (in this issue of Structure) probe the dynamics of processivity clamp proteins from all kingdoms of life. The proteins have similar structures but divergent sequences. Their results show conserved dynamics correlating with primary functions and divergent dynamics for divergent functions.

Predicted disorder-to-order transition mutations in IκBα disrupt function.

IκBα inhibits the transcription factor, NFκB, by forming a very tightly bound complex in which the ankyrin repeat domain (ARD) of IκBα interacts primarily with the dimerization domain of NFκB. The first four ankyrin repeats (ARs) of the IκBα ARD are well-folded, but the AR5-6 region is intrinsically disordered according to amide H/D exchange and protein folding/unfolding experiments. We previously showed that mutations towards the consensus sequence for stable ankyrin repeats resulted in a "prefolded" mutant. To investigate whether the consensus mutations were solely able to order the AR5-6 region, we used a predictor of protein disordered regions PONDR VL-XT to select mutations that would alter the intrinsic disorder towards a more ordered structure (D → O mutants). The algorithm predicted two mutations, E282W and P261F, neither of which correspond to the consensus sequence for ankyrin repeats. Amide exchange and CD were used to assess ordering. Although only the E282W was predicted to be more ordered by CD and amide exchange, stopped-flow fluorescence studies showed that both of the D → O mutants were less efficient at dissociating NFκB from DNA.

Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins.

Amide hydrogen/deuterium exchange detected by mass spectrometry (HXMS) is seeing wider use for the identification of intrinsically disordered parts of proteins. In this review, we discuss examples of how discovery of intrinsically disordered regions and their removal can aid in structure determination, biopharmaceutical quality control, the characterization of how post-translational modifications affect weak structuring of disordered regions, the study of coupled folding and binding, and the characterization of amyloid formation. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring.

EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP. Potential specificity of the two HCPTP variants for tyrosines on EphA2 has not been investigated. We have used a mass spectrometry assay to measure relative rates of dephosphorylation for the two HCPTP variants at phosphotyrosine sites associated with control of the EphA2 kinase activity or interaction with downstream targets. Our results suggest that although both variants dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and HCPTP-B. The SAM domain tyrosine Y960 which has been implicated in downstream PI3K signaling is dephosphorylated exclusively by HCPTP-B. The activation loop tyrosine (Y772) which directly controls kinase activity is dephosphorylated about six times faster by HCPTP-A. In contrast, the juxtamembrane tyrosines (Y575, Y588 and Y594) which are implicated in both control of kinase activity and downstream signaling are dephosphorylated by both variants with similar rates. This difference in preference for dephosphorylation sites on EphA2 not only illuminates the different roles of the two variants of the phosphatase in EphA2 signaling, but also explains why both HCPTP variants are highly conserved in most mammals.

Use of selected reaction monitoring data for label-free quantification of protein modification stoichiometry.

Quantification of protein and PTM abundance in biological samples is an important component of proteomic studies. Label-free methods for quantification using MS are attractive because they are simple to implement and applicable to any experimental system. We demonstrate that PTM stoichiometry can be accurately measured using label-free quantification and selected reaction monitoring. Use of selected reaction monitoring is advantageous with complex biological samples and we show this approach can be used to quantify multiple PTMs independently on a single peptide.

Identification of novel inhibitors for a low molecular weight protein tyrosine phosphatase via virtual screening.

The human cytoplasmic protein tyrosine phosphatase (HCPTP) has been identified as a potential target for inhibition in order to downregulate metastatic transformation in several human epithelial cancers such as breast, prostate and colon cancer. Docking with two scoring functions on both isoforms of HCPTP was employed as an initial virtual screen to identify potential inhibitors. Compounds identified as potential inhibitors via this in silico screen were subjected to kinetic analysis in order to validate their selection as improved inhibitors. Eleven compounds with IC50's of less than 100 microM were identified in a single concentration screen. Five of these compounds were determined to have an IC50 of less than 10 microM; however, all but one of these compounds inhibited via non-specific aggregation. The validated effective inhibitor, which is based on a naphthyl sulfonic acid, strongly resembles a previously synthesized rationally designed azaindole phosphonic acid. This similarity suggests subsequent inhibitor optimization based on this scaffold may generate effective inhibitors of HCPTP. The structural elements of the computationally identified inhibitors are discussed to analyze the combined use of rational design and virtual screening to reduce false negatives in the identification of multiple strong inhibitors of HCPTP.

Tobacco protein separation by aqueous two-phase extraction.

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.