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RAS is the most frequently mutated oncogene in human cancer with nearly ~20% of cancer patients possessing mutations in one of three RAS genes (K, N or HRAS). However, KRAS is mutated in nearly 90% of pancreatic ductal carcinomas (PDAC). Although pharmacological inhibition of RAS has been challenging, KRAS(G12C)-specific inhibitors have recently entered the clinic. While KRAS(G12C) is frequently expressed in lung cancers, it is rare in PDAC. Thus, more broadly efficacious RAS inhibitors are needed for treating KRAS mutant-driven cancers such as PDAC. A RAS-specific tool biologic, NS1 Monobody, inhibits HRAS- and KRAS-mediated signalling and oncogenic transformation both in vitro and in vivo by targeting the α4–α5 allosteric site of RAS and blocking RAS self-association. Here, we evaluated the efficacy of targeting the α4-α5 interface of KRAS as an approach to inhibit PDAC development using an immunocompetent orthotopic mouse model. Chemically regulated NS1 expression inhibited ERK and AKT activation in KRAS(G12D) mutant KPC PDAC cells and reduced the formation and progression of pancreatic tumours. NS1-expressing tumours were characterized by increased infiltration of CD4 + T helper cells. These results suggest that targeting the #x3B1;4-#x3B1;5 allosteric site of KRAS may represent a viable therapeutic approach for inhibiting KRAS-mutant pancreatic tumours.

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain. While Monobody-32 constitutively binds the MLKL hinge region, Monobody-27 binds MLKL via an epitope that overlaps the RIPK3 binding site and is only exposed after phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation. The crystal structures identified two distinct conformations of the MLKL pseudokinase domain, supporting the idea that a conformational transition accompanies MLKL disengagement from RIPK3. These studies provide further evidence that MLKL undergoes a large conformational change upon activation, and identify MLKL disengagement from RIPK3 as a key regulatory step in the necroptosis pathway., Mixed Lineage Kinase Domain-Like (MLKL) pseudokinase is phosphorylated by RIPK3 kinase prior to cell death by necroptosis. Here, the authors use monobodies that bind to the MLKL pseudokinase domain as tools, which allowed them to determine the crystal structures of the MLKL pseudokinase domain in two distinct conformations. By combining their structural data with cell signalling assays and MD simulations they provide evidence that endogenous MLKL preassociates with its upstream regulator RIPK3, and that MLKL disengages from RIPK3 following the induction of necroptosis.

KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favours an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, ‘cross’-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a vital platform to elucidate the structural basis of RAF activation by RAS and to develop newer inhibitors that can disrupt the KRAS dimerization. The methodology developed to specifically probe the intermolecular interactions within KRAS dimer is applicable to many other farnesylated small GTPases.

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

Monobodies, built with the scaffold of the fibronectin type III domain, are among the most well-established synthetic binding proteins. They promote crystallization of challenging molecular systems. They have strong tendency to bind to functional sites and thus serve as drug-like molecules that perturb the biological functions of their targets. Monobodies lack disulfide bonds and thus they are particularly suited as genetically encoded reagents to be used intracellularly. This article reviews recent monobody-enabled studies that reveal new structures, molecular mechanisms and potential therapeutic opportunities. A systematic analysis of the crystal structures of monobody-target complexes suggests important attributes that make monobodies effective crystallization chaperones.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal (Mycoplasma salivarium), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

Graphical abstract, Highlights • Epitope analysis with a designed receptor-binding domain (RBD) mutant of Spike. • Patient antibodies dominantly bind regions outside the ACE2-binding surface of RBD. • Human antibody library sorting for immunogenicity analysis of Spike and RBD. • The RBD is less immunogenic than the other regions of Spike. • The ACE2-binding surface is not a highly immunogenic region within RBD., The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID-19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.

Blocking the interaction between cell-surface receptors and their ligands is a proven therapeutic strategy. Adhesion G protein-coupled receptors (aGPCRs) are key cell-surface receptors that regulate numerous pathophysiological processes, and their large extracellular regions (ECRs) mediate ligand binding and function. The aGPCR GPR56/ADGRG1 regulates central nervous system myelination and melanoma progression by interacting with its ligand, tissue transglutaminase 2 (TG2), but the molecular basis for this interaction is largely undefined. Here, we show that the C-terminal portion of TG2 directly interacted with the GPR56 ECR with high-nanomolar affinity, and used site-directed mutagenesis to identify a patch of conserved residues on the pentraxin/laminin-neurexin-sex-hormone-binding-globulin-like (PLL) domain of GPR56 as the TG2 binding site. Importantly, we also show that the GPR56-TG2 interaction was blocked by previously-reported synthetic proteins, termed monobodies, that bind the GPR56 ECR in a domain- and species-specific manner. This work provides unique tools to modulate aGPCR-ligand binding and establishes a foundation for the development of aGPCR-targeted therapeutics.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.

Summary: RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells. R15 inhibits the signaling and transforming activity of a subset of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., fast exchange mutants). Intracellular expression of R15 reduces the tumor-forming capacity of cancer cell lines driven by select RAS mutants and KRAS(G12D)-mutant patient-derived xenografts (PDXs). Thus, our approach establishes an opportunity to selectively inhibit a subset of RAS mutants by targeting the apo state with drug-like molecules.

Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr–clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications. A synthetic antibody fragment can disrupt the interaction of a GPCR regulatory protein β-arrestin with clathrin, and thereby inhibit induced internalization of GPCRs.

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells., Graphical Abstract Image 1, Highlights • SFK SH2 domains are critical for signaling but difficult to target. • Monobodies bound with high affinity and competed with pY ligand binding. • Discrimination of SH2 domains of the two Src subfamilies was achieved. • Monobody–SH2 complex crystal structures reveal distinct interaction modes. • Monobodies selectively perturbed kinase regulation and downstream signaling.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

Patient mutation–specific BRAF inhibitors alter full-length kinase conformations and promote interactions with GTP-loaded RAS., Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation–containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.

Generation of RAS-targeted therapeutics has long been considered a "holy grail" in cancer research. However, a lack of binding pockets on the surface of RAS and its picomolar affinity for guanine nucleotides have made isolation of inhibitors particularly challenging. We recently described a monobody, termed NS1, that blocks RAS signaling and oncogenic transformation. NS1 binds to the α4-β6-α5 interface of H-RAS and K-RAS thus preventing RAS dimerization and nanoclustering, which in turn prevents RAS-stimulated dimerization and activation of RAF. Interestingly, NS1 reduces interaction of oncogenic K-RAS, but not H-RAS, with RAF and reduces K-RAS plasma membrane localization. Here, we show that these isoform specific effects of NS1 on RAS:RAF are due to the distinct hypervariable regions of RAS isoforms. NS1 inhibited wild type RAS function by reducing RAS GTP levels. These findings reveal that NS1 disrupts RAS signaling through a mechanism that is more complex than simply inhibiting RAS dimerization and nanoclustering.

We have utilized a high-diversity phage display library to engineer antibody fragments (Fabs) that can modulate the activity of the enzyme isocitrate dehydrogenase 1 (IDH1). We show that a conformation-specific Fab can reactivate an IDH1 mutant associated with brain tumors. The results show that this strategy is a first step towards developing “activator drugs” for a large number of genetic disorders where mutations have disrupted protein function.

Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in‐depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid β (Aβ) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid β peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.

Investigations into the genomic landscape of histone modifications in heterochromatic regions have revealed histone H3 lysine 9 dimethylation (H3K9me2) to be important for differentiation and maintaining cell identity. H3K9me2 is associated with gene silencing and is organized into large repressive domains that exist in close proximity to active genes, indicating the importance of maintenance of proper domain structure. Here we show that nickel, a nonmutagenic environmental carcinogen, disrupted H3K9me2 domains, resulting in the spreading of H3K9me2 into active regions, which was associated with gene silencing. We found weak CCCTC-binding factor (CTCF)-binding sites and reduced CTCF binding at the Ni-disrupted H3K9me2 domain boundaries, suggesting a loss of CTCF-mediated insulation function as a potential reason for domain disruption and spreading. We furthermore show that euchromatin islands, local regions of active chromatin within large H3K9me2 domains, can protect genes from H3K9me2-spreading-associated gene silencing. These results have major implications in understanding H3K9me2 dynamics and the consequences of chromatin domain disruption during pathogenesis.

Variability in the quality of antibodies to histone post-translational modifications (PTMs) presents widely recognized hindrance in epigenetics research. Here, by using antibody engineering technologies we produced recombinant antibodies directed to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation. These recombinant antibodies performed well in common epigenetics applications, and their high specificity enabled us to identify positive and negative correlations among histone PTMs.

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer’s disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ∼18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.

The two isoforms of the Bcr-Abl tyrosine kinase, p210 and p190, are associated with different leukemias and have a dramatically different signaling network, despite similar kinase activity. To provide a molecular rationale for these observations, we study the Dbl-homology (DH) and Pleckstrin-homology (PH) domains of Bcr-Abl p210, which constitute the only structural differences to p190. Here we report high-resolution structures of the DH and PH domains and characterize conformations of the DH–PH unit in solution. Our structural and functional analyses show no evidence that the DH domain acts as a guanine nucleotide exchange factor, whereas the PH domain binds to various phosphatidylinositol-phosphates. PH-domain mutants alter subcellular localization and result in decreased interactions with p210-selective interaction partners. Hence, the PH domain, but not the DH domain, plays an important role in the formation of the differential p210 and p190 Bcr-Abl signaling networks., The Bcr-Abl tyrosine kinases p210 and p190 are linked to different leukemias and differ by the Dbl homology (DH) and Pleckstrin-homology (PH) domains. Here the authors characterize structures of the Bcr-Abl p210 DH and PH domains and find that the PH domain is important for the cellular localization and signaling network of p210.

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

Adhesion G protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we termPentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development invivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology.

The γ-secretase complex, composed of presenilin, anterior-pharynx-defective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformation-specific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245–630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain ( D YIGS a nd p eptidase; residues 261–502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a “substrate receptor” within the γ-secretase complex.

SummaryChronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.PaperFlick

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the α-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an α-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

Objective To examine the relationship between an antibody against GAPDH-2, a sperm-specific protein, and infertility of female mice. Design Basic research. Setting National Research Institute for Family Planning Beijing, World Health Organization Collaboration Center of Human Reproduction. Animal(s) New Zealand rabbit, NIH and ICR mice. Intervention(s) None. Main Outcome Measure(s) Enzyme-linked immunoabsorbent assay, Western blot and indirect immunostaining assays, standard fertility assay, and sperm agglutination assay. Result(s) Antibodies against the full-length GAPDH-2 were raised. Its specificity was assessed by immunoblotting and indirect immunostaining assays. The antibody immunoreacted with human sperm GAPDH-2 and the mouse homolog GAPDS but did not cross-react with GAPDH. Treatment of female mice with IP injection of anti-GAPDH-2 serum significantly reduced their fertility. Anti-GAPDH-2 serum caused the agglutination of normal mice sperm in vitro. The anti-GAPDH-2 antibody was detectable in the sera and uterine fluid of the mice immunized with GAPDH-2. Conclusion(s) These results show that GAPDH-2 should be further evaluated as a promising candidate in the development of an antifertility immunogen. Detecting anti-GAPDH-2 antibodies in the bodily fluid of subjects afflicted with indeterminate infertility may be a new diagnostic index.

The preparation of diffraction quality crystals remains the major bottleneck in macromolecular X-ray crystallography. A crystallization chaperone is an auxiliary protein, such as fragments of monoclonal antibodies, that binds to and increases the crystallization probability of a target molecule of interest. Such chaperones reduce conformational heterogeneity, mask counterproductive surfaces while extending surfaces predisposed to forming crystal contacts, and provide phasing information. Crystallization chaperones generated using recombinant technologies have emerged as superior alternatives that increase the throughput and eliminate inherent limitations associated with antibody production by animal immunization and the hybridoma technology.

Rhomboid family members are widely conserved and found in all three kingdoms of life. They are serine proteases and serve important regulatory functions. In the present study, a novel gene highly expressed in the testis, RHBDD1, is shown to be a new member of the Rhomboid family, participating in the cleavage of BIK, a proapoptotic member of the Bcl-2 family. The RHBDD1-involved proteolytic modification is upstream of the BIK protein degradation pathway. Mutagenesis studies show that the amino acid residues glycine142 and serine144 of RHBDD1 are crucial for its activity in cleaving BIK at a site located in the transmembrane region. Overexpression or knock-down of RHBDD1 in HEK 293T cells can reduce or enhance BIK-mediated apoptosis, respectively. The present findings suggest that, by acting as a serine protease, RHBDD1 modulates BIK-mediated apoptotic activity.

Stability evaluation of many mutants can lead to a better understanding of the sequence determinants of a structural motif and of factors governing protein stability and protein evolution. The traditional biophysical analysis of protein stability is low throughput, limiting our ability to widely explore sequence space in a quantitative manner. In this study, we have developed a high-throughput library screening method for quantifying stability changes, which is based on protein fragment reconstitution and yeast surface display. Our method exploits the thermodynamic linkage between protein stability and fragment reconstitution and the ability of the yeast surface display technique to quantitatively evaluate protein-protein interactions. The method was applied to a fibronectin type III (FN3) domain. Characterization of fragment reconstitution was facilitated by the co-expression of two FN3 fragments, thus establishing a yeast surface two-hybrid method. Importantly, our method does not rely on competition between clones and thus eliminates a common limitation of high-throughput selection methods in which the most stable variants are recovered predominantly. Thus, it allows for the isolation of sequences that exhibit a desired level of stability. We identified more than 100 unique sequences for a beta-bulge motif, which was significantly more informative than natural sequences of the FN3 family in revealing the sequence determinants for the beta-bulge. Our method provides a powerful means for the rapid assessment of the stability of many variants, for the systematic assessment of the contribution of different factors to protein stability, and for enhancement of the protein stability.

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This "antigen clasping" produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody-antigen recognition and suggests a strategy for developing extremely specific antibodies.

High degrees of sequence and conformation complexity found in natural protein interaction interfaces are generally considered essential for achieving tight and specific interactions. However, it has been demonstrated that specific antibodies can be built by using an interface with a binary code consisting of only Tyr and Ser. This surprising result might be attributed to yet undefined properties of the antibody scaffold that uniquely enhance its capacity for target binding. In this work we tested the generality of the binary-code interface by engineering binding proteins based on a single-domain scaffold. We show that Tyr/Ser binary-code interfaces consisting of only 15–20 positions within a fibronectin type III domain (FN3; 95 residues) are capable of producing specific binding proteins (termed “monobodies”) with a low-nanomolar K d . A 2.35-Å x-ray crystal structure of a monobody in complex with its target, maltose-binding protein, and mutation analysis revealed dominant contributions of Tyr residues to binding as well as striking molecular mimicry of a maltose-binding protein substrate, β-cyclodextrin, by the Tyr/Ser binary interface. This work suggests that an interaction interface with low chemical diversity but with significant conformational diversity is generally sufficient for tight and specific molecular recognition, providing fundamental insights into factors governing protein–protein interactions.

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.

hSMP-1 is a human sperm membrane protein expressed during development. It is a testis-specific component produced during male germ cell differentiation. Proteins that interact with hSMP-1 were identified by the application of the yeast two-hybrid system. One of the components, RanBPM, was found to be associated with hSMP-1 under both in vitro and in vivo conditions. In the human testis, RanBPM is produced in spermatogonia and primary spermatocytes, suggesting expression during the early stages of spermatogenesis; whereas in the rat testis, it is located in round and elongated spermatids, similar to hSMP-1, suggesting expression of both components during spermiogenesis. Images obtained by immunofluorescence and confocal scanning microscopy of CHO-K1 cells co-transfected with pEGFP-C1-hSMP-1 and pDsRed1-Nl-RanBPM revealed that RanBPM and hSMP-1 are distributed in discrete loci throughout the cytoplasm. When superimposed, the stained spots appeared as congruent yellow areas, indicative of co-localization and probable complex formation of these two components. This interaction between hSMP-1 and RanBPM may be involved in the process of male germ cell differentiation. In CHO-Kl cells transfected with pEGFP-Cl-hSMP-1, the exogenously expressed hSMP-1 was found to co-localize with alpha-tubulin. Depolymerization of microtubules can be induced in CHO-Kl cells by cold treatment. In cells transfected with the pEGFP-Cl vector, the dispersed tubulins promptly reassembled upon warming. However, in cells transfected with pEGFP-Cl-hSMP-1, reassembly of the dispersed tubulins was blocked even upon rewarming of the cells. These findings suggest that hSMP-1 interacts with tubulins and thereby may modulate microtubule assembly and/or activity.

We investigated mechanical unfolding of Borrelia burgdorferi outer surface protein A (OspA), a Lyme disease antigen containing a unique single-layer beta-sheet, with atomic force microscopy (AFM). We mechanically stretched a monomeric unit, rather than a tandem repeat, by pulling it from its N and C-terminal residues without using intervening polymer as a spacer. We detected two peaks in the force-extension profile before the final rupture of a fully extended polypeptide, which we interpreted as unfolding of multiple substructures in OspA. The double-peaked unfolding curves are consistent with results of previous thermodynamic studies showing two cooperative units in OspA. The mechanical unfolding processes were reversible, and the two substructures refolded within one second. Mutations near the boundary of the two thermodynamic cooperative units reduced the height of the first unfolding peak to undetectable levels and marginally affected the second one, indicating that the boundary between the two mechanical substructures is related to that previously assigned between the thermodynamic cooperative units. Based on a "worm-like chain" analysis of our AFM data, we propose a model for mechanical unfolding of OspA, where nearly a half of the chain is stretched with minimal resistive force, followed by sequential breakdown of C-terminal and N-terminal substructures. Based on these results, we discuss similarities and differences between mechanical and thermodynamic unfolding reactions of OspA. This work demonstrates that AFM study of monomeric proteins can elucidate details of the intramolecular mechanics of protein substructures.

The expression of stage-specific genes during spermatogenesis was determined by isolating two segments of rat seminiferous tubule at different stages of the germinal epithelium cycle delineated by transillumination-delineated microdissection, combined with differential display polymerase chain reaction to identify the differential transcripts formed. A total of 22 cDNAs were identified and accepted by GenBank as new expressed sequence tags. One of the expressed sequence tags was radiolabeled and used as a probe to screen a rat testis cDNA library. A novel full-length cDNA composed of 2228 bp, designated as RSD-3 (rat sperm DNA no.3, GenBank accession no. AF094609) was isolated and characterized. The reading frame encodes a polypeptide consisting of 526 amino acid residues, containing a number of DNA binding motifs and phosphorylation sites for PKC, CK-II, and p34cdc2. Northern blot of mRNA prepared from various tissues of adult rats showed that RSD-3 is expressed only in the testis. The initial expression of the RSD-3 gene was detected in the testis on the 30th postnatal day and attained adult level on the 60th postnatal day. Immunolocalization of RSD-3 in germ cells of rat testis showed that its expression is restricted to primary spermatocytes, undergoing meiosis division I. A human testis homologue of RSD-3 cDNA, designated as HSD-3.1 (GenBank accession no. AF144487) was isolated by screening the Human Testis Rapid-Screen arrayed cDNA library panels by RT-PCR. The exon-intron boundaries of HSD-3.1 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The cDNA consisted of 12 exons that span approximately 52.8 kb of the genome sequence and was mapped to chromosome 14q31.3.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.

It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions.

The human estrogen receptor-α (hER) is a ligand-activated transcription factor that functions as a homodimer. We sought to further understand the molecular processes involved in dimerization, and to develop a reagent that may function as an antiestrogen independent of the ligand binding site. To this end, we designed a 16-residue ‘dimer-interface’ oligopeptide derived from the helical region of the hER which is directly involved in dimerization. This peptide, termed the I-box peptide, has a high helical propensity in aqueous solution. The I-box peptide blocks hER action by causing aggregation and precipitation of both the ligand-bound and apo-hER. This effect is dependent on the helical nature of the peptide. A single Ile to Pro mutation in the helical region of the I-box peptide significantly reduces the helical content and abolishes the precipitation activity. Furthermore, the peptide activity appears to be specific for the hER. The I-box peptide does not significantly affect other proteins or steroid receptors tested. A homologous peptide derived from the nuclear receptor RXRα dimer interface, and a LXXLL-containing peptide from the coactivator TIF2 have no detectable in vitro effect on hER function or solubility. Our data suggest that rationally designed molecules capable of affecting steroid receptor quaternary structures may be potential avenues for the development of specific inhibitors of this class of proteins.

The YWK-II protein in human spermatozoa is structurally related to the betaA4-amyloid precursor protein of Alzheimer disease and has high similarity with amyloid precusor homologues. Antibodies to the YWK-II protein agglutinate human spermatozoa and may be a potential cause of infertility. In the present study, a yeast two-hybrid system (MATCHMAKER Two-Hybrid System 2; Clontech, Palo Alto, CA) was used to screen a rat ovary cDNA library for potential ligands capable of interacting with the YWK-II component. Mullerian-inhibiting substance was found to interact with the extracellular domain of YWK-II protein. The interaction was confirmed by a binding experiment in vitro and surface plasmon resonance assays. The recombinant Mullerian-inhibiting substance can significantly increase the viability and longevity of human spermatozoa after 5 and 22 h of incubation, presumably through binding the YWK-II component on the sperm membrane. The results of this study indicate that the YWK-II sperm membrane protein may function as a receptor for Mullerian-inhibiting substance.

An 80-kDa human sperm antigen (80-kDa HSA) has been identified as a sperm protein responsible for inducing immunoinfertility. Immunization with the purified protein induced infertility in male and female rats. Immunohistochemical and immunofluorescent studies have demonstrated that the antigen is specific to spermatozoa. The present study describes the partial amino acid sequencing of 80-kDa HSA. The homogeneous protein was electrophoretically transferred onto a PVDF membrane and the excised band of 80-kDa HSA was used to determine the partial N-terminal amino acid sequence. The protein was then subjected to enzymatic digestion with endoproteinase Lys-C and endoproteinase Glu-C. The partial amino acid sequence of the major peptides thus obtained was determined. The digestion with endoproteinase Lys-C generated 4 major peptides, two of which showed partial sequence homology with lactoferrin. Endoproteinase Glu-C digestion produced 3 major peptides. The sequences of the 2 peptides were determined for which no matches were found in the databank. These results confirmed earlier observations that 80-kDa HSA is a sperm-specific protein that is chemically distinct from any other protein involved in normal physiological process. Earlier studies have demonstrated that it is antigenic, efficacious, conserved, and could be a promising candidate for the development of an antifertility vaccine.

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.