The fibronectin type III domain (FN3) has become one of the most widely used non-antibody scaffolds for generating new binding proteins. Because of its structural homology to the immunoglobulin domain, combinatorial libraries of FN3 designed to date have primarily focused on introducing amino acid diversity into three loops that are equivalent to antibody complementarity-determining regions. Here, we report an FN3 library that utilizes alternative positions for presenting amino acid diversity. We diversified positions on a β-sheet and surface loops that together form a concave surface. The new library produced binding proteins (termed “monobodies”) to multiple target proteins, generally with similar efficacy as the original, loop-focused library. The crystal structure of a monobody generated from the new library in complex with its target, the Abl SH2 domain, revealed that a concave surface of the monobody, as intended in our design, bound to a convex surface of the target with the interface area being among the largest of published structures of monobody-target complexes. This mode of interaction differs from a common binding mode for single-domain antibodies and antibody mimics in which recognition loops recognize clefts in targets. Together, this work illustrates the utilization of different surfaces of a single immunoglobulin-like scaffold to generate binding proteins with distinct characteristics.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

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.M16 metalloprotease | X-ray crystallography | substrate recognition P roteins in living organisms face acute and chronic challenges to their integrity, which necessitate proteostatic processes to protect their functions (1). Protein-protease networks play a key role in proteostasis by ensuring proper protein function through protein turnovers (2). Amyloidogenic peptides, such as amyloid β (Aβ) and amylin, present a major challenge to proteostasis, because they can form toxic aggregates that impair diverse physiological functions and contribute to human diseases (3, 4). Insulin-degrading enzyme (IDE), a Zn 2+ -metalloprotease, prefers to degrade amyloidogenic peptides to prevent the formation of amyloid fibrils (3). Exemplary substrates of IDE are insulin and Aβ, which are critical for the development of type 2 diabetes mellitus (DM2) and Alzheimer's disease (AD), respectively. Genetic analyses strongly support functional roles of IDE in the clearance of insulin and Aβ (2, 3). In humans, several single nucleotide polymorphisms at the IDE locus on human chromosome 10q are associated with DM2 and late-onset AD (5, 6).Structural analyses have provided significant insights to substrate recognition and catalysis by IDE. IDE has two ∼50-kDa αβαβα N-terminal (IDE-N) and C-terminal (IDE-C) halves, which are linked by a short hinge loop...

The γ-secretase complex, composed of presenilin, anterior-pharynxdefective 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 conformationspecific 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 (DYIGS and peptidase; 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.A lzheimer's disease (AD), a progressive neurodegenerative disease, is the most prevalent cause of dementia in humans. The principal neuropathological hallmark of AD is the presence of senile plaques composed of dystrophic neurites surrounding extracellular aggregates of Aβ peptides (1). Aβ peptides are liberated from amyloid precursor proteins (APP) by the concerted action of β-site APP cleaving enzyme 1 and γ-secretase (2, 3). γ-Secretase is a macromolecular complex consisting of presenilin 1 or presenilin 2 (PS1 or PS2), anterior-pharynx-defective 1 (APH-1), nicastrin (NCT), and presenilin enhancer 2 (PEN-2) that catalyzes intramembranous proteolysis of several membrane-tethered substrates (4). Evidence has emerged to reveal the functions of each subunit: PS is the catalytic subunit (5); APH-1 serves as a scaffold for the complex assembly (6); NCT seems to be responsible for substrate binding (7); and PEN-2 promotes endoproteolytic cleavage of PS and "activation" of the enzyme complex (5, 8). Although γ-secretase cleaves a multiplicity of substrates at heterogeneous sites within individual transmembrane domains, the molecular mechanism(s) underlying substrate recognition and processing remain elusive.NCT, a 709-aa type I transmembrane glycoprotein with a large, heavily glycosylated ectodomain (ECD), was first identified as a PS-inter...

Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (KD ~ 10 nM) for the Fc region of the IgG, but a much lower affinity (KD ~ low εM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 10,000-fold change in affinity from pH 7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.