Minimal Binding Domain Research Articles

Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors.

Parvovirus B19 (B19V) infects human erythroid progenitor cells (EPCs) and causes several hematological disorders and fetal hydrops. Amino acid (aa) 5-68 of minor capsid protein VP1 (VP1u5-68aa) is the minimal receptor binding domain for B19V to enter EPCs. Here, we carried out a genome-wide CRISPR-Cas9 guide RNA screen and identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. AXL gene silencing in ex vivo expanded EPCs remarkably decreased B19V internalization and replication. Additions of the recombinant AXL extracellular domain or a polyclonal antibody against it upon infection efficiently inhibited B19V infection of ex vivo expanded EPCs. Moreover, B19V VP1u interacted with the recombinant AXL extracellular domain in vitro at a relatively high affinity (KD=103 nM). Collectively, we provide evidence that AXL is a co-receptor for B19V infection of EPCs.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

Predicting the Effect of Mutations in the KRas/c-Raf-RBD Protein-Protein Interface

KRas is a small GTPase commonly implicated in several difficult-to-treat cancers such as pancreatic ductal adenocarcinoma (PDAC). KRas normally cycles between an active, GTP-bound form and an inactive, GDP-bound form. Active KRas functions by forming protein-protein interactions (PPIs) with multiple effector proteins in order to regulate various important signal transduction pathways. However, when KRas is mutated it is constitutively active which leads to signal transduction pathway dysregulation that subsequently increases and sustains tumorigenicity and invasiveness. KRas has long been considered an “undruggable” target due to its picomolar affinity for its substrate. However, blocking the PPIs between KRas and its effectors eliminates harmful downstream effects. The tightest known binder of KRas is c-Raf, an enzyme in the ERK1/2 pathway. The Ras-binding domain (RBD) is the minimal binding domain of c-Raf that selectively binds to active, GTP-bound KRas. Previous work has measured Kd for various mutations in the KRas/c-Raf-RBD interface [1, 2]. We use OSPREY [3] (Open Source Protein REdesign for You), a state-of-the-art software package for computational structure-based protein design (CSPD), along with K∗ [4], an algorithm that estimates the binding constant for a given protein complex, to computationally predict the effect of these mutations. We compared our computational predictions to the experimental measurements and found that we can accurately predict the effect of these mutations. These results validate the accuracy of CSPD with OSPREY to target protein-protein interfaces and give us confidence that we can accurately redesign the KRas/c-Raf-RBD interface for future work towards targeting “undruggable” proteins. [1] M. Fridman, et al. Journal of Biological Chemistry, 275(39):30363-30371, 2000. [2] C. Kiel, et al. Journal of Biological Chemistry, 284(46):31893-31902, 2009. [3] P. Gainza, et al. Methods in enzymology, 523:87-107, 2013. [4] I. Georgiev, et al. Journal of computational chemistry, 29(10):1527-1542, 2008.

Facilitating identification of minimal protein binding domains by cross-linking mass spectrometry

Characterization of protein interaction domains is crucial for understanding protein functions. Here we combine cross-linking mass spectrometry (XL-MS) with deletion analysis to accurately locate minimal protein interaction domains. As a proof of concept, we investigated in detail the binding interfaces of two protein assemblies: the complex formed by MICAL3, ELKS and Rab8A, which is involved in exocytosis, and the complex of SLAIN2, CLASP2 and ch-TOG, which controls microtubule dynamics. We found that XL-MS provides valuable information to efficiently guide the design of protein fragments that are essential for protein interaction. However, we also observed a number of cross-links between polypeptide regions that were dispensable for complex formation, especially among intrinsically disordered sequences. Collectively, our results indicate that XL-MS, which renders distance restrains of linked residue pairs, accelerates the characterization of protein binding regions in combination with other biochemical approaches.

Abstract IA14: Engaging T cells against cancer: BLINCYTO® and beyond – the BiTE® platform

Abstract T-cell-based cancer immunotherapies are at the forefront of a new era in cancer treatment. Engaging T cells through BiTE® (bispecific T-cell engager) antibody constructs is one of the most advanced immunotherapeutic approaches. BiTE® antibody constructs have a unique design and mechanism of action. They are constructed by genetically linking the minimal binding domains of monoclonal antibodies for a tumor-associated surface antigen and for the T-cell receptor-associated molecule CD3 onto a single polypeptide chain. Concurrent engagement of the target cell antigen and CD3 leads to activation of polyclonal cytotoxic T-cells, resulting in target cell lysis. The first BiTE®, blinatumomab, targeting CD19 and CD3, is being investigated in a broad range of B-cell malignancies and has recently received an accelerated approval by the US FDA for Philadelphia chromosome-negative relapsed/refractory B-acute lymphoblastic leukemia under the trade name BLINCYTO®. In addition, several BiTE® antibody constructs targeting other tumor antigens have entered the clinical stage of development. Most recent updates about the BiTE® platform will be presented. Citation Format: Dirk Nagorsen. Engaging T cells against cancer: BLINCYTO® and beyond – the BiTE® platform. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA14.

Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22

Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1–58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140–162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a “nutcracker” with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.

Soluble Notch ligand and receptor peptides act antagonistically during angiogenesis.

Notch signalling is essential for blood vessel formation. During angiogenesis, the Notch ligand DLL4 on the leading tip cell activates Notch receptors on the adjacent stalk cells. DLL4-Notch signalling is impaired by the Notch ligand JAG1 in endothelial cells. The Delta/Serrate/Lag2 (DSL) domain of the Notch ligands binds to the EGF-like repeats 11-13 of the Notch receptor. This study aimed to elucidate how soluble proteins containing these short domains interfere with Notch signalling during angiogenesis. Adenoviral vectors were generated to express the DSL domains of DLL1, DLL4, JAG1, and the Notch1 EGF-like repeats 11-13 fused to immunoglobulin-G heavy chain. These soluble ligand peptides inhibited Notch signalling in endothelial cells and this caused hyperbranching in cellular angiogenesis assays and in the neonatal mouse retina. The soluble Notch receptor peptides bound stronger to JAG1 than DLL4 ligands, resulting in increased signalling activity. This led to impaired tip cell formation and less vessel sprouting in the retina. The minimal binding domains of Notch ligands are sufficient to interfere with Notch signalling. The corresponding soluble Notch1 EGF11-13 peptide binds stronger to inhibitory Notch ligands and thereby promotes Notch signalling in endothelial cells.

Defining the Interactions and Role of DCAF1/VPRBP in the DDB1-Cullin4A E3 Ubiquitin Ligase Complex Engaged by HIV-1 Vpr to Induce a G2 Cell Cycle Arrest

HIV viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD). Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

1H, 13C, and 15N chemical shift assignments for the complex of calmodulin with minimal binding domain from HIV-1 matrix protein

1H, 13C, and 15N chemical shift assignments for the complex of calmodulin with minimal binding domain from HIV-1 matrix protein

Shootin1 Acts in Concert with KIF20B to Promote Polarization of Migrating Neurons

Shootin1 has been ascribed a role in regulating polarization of primary hippocampal neurons. To better understand the possible role of Shootin1 in the developing brain, we identified a member of the kinesin superfamily, KIF20B, as a novel Shootin1 interacting protein and a potential mediator of Shootin1 interaction with microtubules. KIF20B/Shootin1 binding was mapped to a 57 aa KIF20B sequence, which was used as a dominant-negative fragment. Direct interaction between that peptide (MBD) and Shootin1 was confirmed by surface plasmon resonance-based technology and the affinity was determined in the 10⁻⁷ m range. The proteins are expressed in the developing brain and formed a complex in vivo based on coimmunoprecipitation experiments and coimmunostaining in primary neurons. In primary hippocampal neurons Kif20b knockdown reduced Shootin1 mobilization to the developing axon, as evidenced by immunostaining and fluorescence recovery after photobleaching analysis, suggesting that Shootin1 is a novel KIF20B cargo. shRNA targeting of Shootin1 reduced PIP3 accumulation in the growth cone, as did Kif20b shRNA. In the developing mouse brain, Kif20b knockdown or expression of the KIF20B minimal binding domain inhibited neuronal migration, and in vivo migration assays suggested that Shootin1/Kif20b acts in the same genetic pathway. Time-lapse imaging of multipolar cells in the subventricular zone revealed that downregulating levels of either Shootin1 or Kif20b hindered the transition from multipolar to bipolar cells. Collectively, our data demonstrate the importance of the Shootin1/KIF20B interaction to the dynamic process of pyramidal neuronal polarization and migration.

The N-terminal tetra-peptide (IPDE) short extension of the U-box motif in rice SPL11 E3 is essential for the interaction with E2 and ubiquitin-ligase activity

Rice, a monocot model plant, contains at least 77 U-box E3 ubiquitin (Ub)-ligases and 48 E2 Ub-conjugating enzymes. Here, we investigated the minimal binding domain of rice SPL11 U-box E3 to its E2 partners. Serial deletions and site-directed mutagenesis analyses indicated that, in addition to an intact U-box motif, the N-terminal tetra-peptide (IPDE) short extension of the U-box was essential for the interaction of SPL11 with E2s and Ub-ligase activity. The Ile and Pro residues at the –4 and –3 positions of the U-box, respectively, were crucial for this interaction. These results suggest that the N-terminal tetra-peptide extension of the U-box participates in the specific interaction of SPL11 E3 with E2s in a sequence-specific manner in rice.

Substrate-selective and Calcium-independent Activation of CaMKII by α-Actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.

Plasmodium falciparum ‐induced CD36 clustering rapidly strengthens cytoadherence via p130CAS‐mediated actin cytoskeletal rearrangement

The adhesion of infected red blood cells (IRBCs) to microvascular endothelium is critical in the pathogenesis of severe malaria. Here we used atomic force and confocal microscopy to examine the adhesive forces between IRBCs and human dermal microvascular endothelial cells. Initial contact of the cells generated a mean ± sd adhesion force of 167 ± 208 pN from the formation of single or multiple bonds with CD36. The strength of adhesion increased by 5- to 6-fold within minutes of contact through a signaling pathway initiated by CD36 ligation by live IRBCs, or polystyrene beads coated with anti-CD36 or PpMC-179, a recombinant peptide representing the minimal binding domain of the parasite ligand PfEMP1 to CD36. Engagement of CD36 led to localized phosphorylation of Src family kinases and the adaptor protein p130CAS, resulting in actin recruitment and CD36 clustering by 50-60% of adherent beads. Uninfected red blood cells or IgG-coated beads had no effect. Inhibition of the increase in adhesive strength by the Src family kinase inhibitor PP1 or gene silencing of p130CAS decreased adhesion by 39 ± 12 and 48 ± 20%, respectively, at 10 dyn/cm(2) in a flow chamber assay. Modulation of adhesive strength at PfEMP1-CD36-actin cytoskeleton synapses could be a novel target for antiadhesive therapy.

NMR, Biophysical, and Biochemical Studies Reveal the Minimal Calmodulin Binding Domain of the HIV-1 Matrix Protein

Subcellular distribution of Calmodulin (CaM) in human immunodeficiency virus type-1 (HIV-1)-infected cells is distinct from that observed in uninfected cells. CaM has been shown to interact and co-localize with the HIV-1 Gag protein in infected cells. However, the precise molecular mechanism of this interaction is not known. Binding of Gag to CaM is dependent on calcium and is mediated by the N-terminal-myristoylated matrix (myr(+)MA) domain. We have recently shown that CaM binding induces a conformational change in the MA protein, triggering exposure of the myristate group. To unravel the molecular mechanism of CaM-MA interaction and to identify the minimal CaM binding domain of MA, we devised multiple approaches utilizing NMR, biochemical, and biophysical methods. Short peptides derived from the MA protein have been examined. Our data revealed that whereas peptides spanning residues 11-28 (MA-(11-28)) and 31-46 (MA-(31-46)) appear to bind preferentially to the C-terminal lobe of CaM, a peptide comprising residues 11-46 (MA-(11-46)) appears to engage both domains of CaM. Limited proteolysis data conducted on the MA-CaM complex yielded a MA peptide (residues 8-43) that is protected by CaM and resistant to proteolysis. MA-(8-43) binds to CaM with a very high affinity (dissociation constant = 25 nm) and in a manner that is similar to that observed for the full-length MA protein. The present findings provide new insights on how MA interacts with CaM that may ultimately help in identification of the functional role of CaM-Gag interactions in the HIV replication cycle.

Extending Half-life by Indirect Targeting of the Neonatal Fc Receptor (FcRn) Using a Minimal Albumin Binding Domain

The therapeutic and diagnostic efficiency of engineered small proteins, peptides, and chemical drug candidates is hampered by short in vivo serum half-life. Thus, strategies to tailor their biodistribution and serum persistence are highly needed. An attractive approach is to take advantage of the exceptionally long circulation half-life of serum albumin or IgG, which is attributed to a pH-dependent interaction with the neonatal Fc receptor (FcRn) rescuing these proteins from intracellular degradation. Here, we present molecular evidence that a minimal albumin binding domain (ABD) derived from streptococcal protein G can be used for efficient half-life extension by indirect targeting of FcRn. We show that ABD, and ABD recombinantly fused to an Affibody molecule, in complex with albumin does not interfere with the strictly pH-dependent FcRn-albumin binding kinetics. The same result was obtained in the presence of IgG. An in vivo study performed in rat confirmed that the clinically relevant human epidermal growth factor 2 (HER2)-targeting Affibody molecule fused to ABD has a similar half-life and biodistribution profile as serum albumin. The proof-of-concept described may be broadly applicable to extend the in vivo half-life of short lived biological or chemical drugs ultimately resulting in enhanced therapeutic or diagnostic efficiency, a more favorable dosing regimen, and improved patient compliance.

The role of His-50 of α-synuclein in binding Cu(ii): pH dependence, speciation, thermodynamics and structure

Copper interaction with alpha synuclein (αS) has been shown to accelerate aggregation and oligomerization of the protein. Three different αS copper binding domains have been proposed: (i) the N-terminal residues (1-9) that represent the minimal copper binding domain; (ii) the His-50 imidazole and (iii) the Asp and Glu residues within the acidic C-terminal domain. The copper coordination at the N-terminus has been extensively characterized and it is generally accepted that it provides the highest affinity site. The same does not hold for the role played by His-50 in copper binding. In this work Cu(ii) coordination to peptide fragments encompassing residues 45-55 of αS has been exhaustively characterized, including systems containing the inherited mutations E46K and A53T, as model peptides of the His-50 site. Through potentiometric titrations all the speciation profiles have been determined and the stability constants have been used to estimate the dissociation constants of complexes corresponding to the binding modes at pH 6.5 and 7.5. Spectroscopic analyses allowed determination of (i) the copper coordination sphere, (ii) its geometry and (iii) the constraints wherefrom the 3D structural models of the copper complexes could be obtained.

Kinesin-1 plays a role in transport of SNAP-25 to the plasma membrane

The cellular molecular motor kinesin-1 mediates the microtubule-dependent transport of a range of cargo. We have previously identified an interaction between the cargo-binding domain of kinesin-1 heavy chain KIF5B and the membrane-associated SNARE proteins SNAP-25 and SNAP-23. In this study we further defined the minimal SNAP-25 binding domain in KIF5B to residues 874–894. Overexpression of a fragment of KIF5B (residues 594–910) resulted in significant colocalization with SNAP-25 with resulting blockage of the trafficking of SNAP-25 to the periphery of cells. This indicates that kinesin-1 facilitates the transport of SNAP-25 containing vesicles as a prerequisite to SNAP-25 driven membrane fusion events.

Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38

Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor alpha subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor alpha regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidyl-prolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn(2+) finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well as regulation of PHD2 protein abundance by FKBP38, is dependent on membrane- anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically our data indicate that PHD2 protein stability is regulated by a ubiquitin-independent proteasomal pathway involving FKBP38 as adaptor protein that mediates proteasomal interaction. We hypothesize that FKBP38-bound PHD2 is constantly degraded whereas cytosolic PHD2 is stable and able to function as an active prolyl-4-hydroxylase.

Synthesis and characterization of a high-affinity αvβ6-specific ligand for in vitro and in vivo applications

The α(v)β(6) integrin is an attractive therapeutic target for several cancers due to its role in metastasis and its negligible expression in normal tissues. We previously identified a peptide from a phage-displayed peptide library that binds specifically to α(v)β(6). The tetrameric version of the peptide has higher affinity for its cellular targets than the corresponding monomers. However, the inefficient synthesis limits its clinical potential. We report here a convergent synthesis producing the tetrameric peptide in high yield and purity. The ease of the synthesis allows for rapid optimization of the peptide. We have optimized this α(v)β(6) integrin-binding peptide, determining the minimal binding domain and valency. Importantly, the half-maximal binding affinity of the optimal peptide for its target cell is in the 40 to 60 pmol/L range, rivaling the affinity of commonly used antibody-targeting reagents. This peptide mediates cell-specific uptake, is functional in diagnostic formats, is stable in sera, and can home to a tumor in an animal. We anticipate that this high-affinity ligand for α(v)β(6) will find clinical use as a diagnostic and therapeutic reagent.