The discovery and evaluation of [18F]BMS-986229, a novel macrocyclic peptide PET radioligand for the measurement of PD-L1 expression and in-vivo PD-L1 target engagement.

PURPOSE: A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1. METHODS: [18F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression. RESULTS: Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%. CONCLUSION: A novel 18F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [18F]BMS-986229 to measure PD-L1 expression in tumors.

Identification of Inhibitors of the Disease-Associated Protein Phosphatase Scp1 Using Antibody Mimetic Molecules.

Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1.

Structure determination and analysis of titin A-band fibronectin type III domains provides insights for disease-linked variants and protein oligomerisation.

Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies.

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.

Mutational and biophysical robustness in a prestabilized monobody.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium.

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.

Auto- and Hetero-Catalytic Processing of the N-Terminal Propeptide Promotes the C-Terminal Fibronectin Type III Domain-Mediated Dimerization of a Thermostable Vpr-like Protease.

Microbial Vpr-like proteases are extracellular multidomain subtilases with diverse functions and can form oligomers, but their maturation and oligomerization mechanisms remain to be elucidated. Here, we report a novel Vpr-like protease (BTV) from thermophilic bacterium Brevibacillus sp. WF146. The BTV precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain with an inserted protease-associated (PA) domain, two tandem fibronectin type III domains (Fn1 and Fn2), and a C-terminal propeptide. The BTV proform (pro-BTV) could be autoprocessed into the mature form (mBTV) via two intermediates lacking the N- or C-terminal propeptide, respectively, and the C-terminal propeptide delays the autocatalytic maturation of the enzyme. By comparison, pro-BTV is more efficiently processed into mBTV by protease TSS from strain WF146. Purified mBTV is a Ca2+-dependent thermostable protease, showing optimal activity at 60°C and retaining more than 60% of activity after incubation at 60°C for 8 h. The PA domain is important for enzyme stability and contributes to the substrate specificity of BTV by restricting the access of protein substrates to the active site. The proform and mature form of BTV exist as a monomer and a homodimer, respectively, and the dimerization is mediated by the Fn1 and Fn2 domains. The N-terminal propeptide of BTV not only acts as intramolecular chaperone and enzymatic inhibitor but also inhibits the homodimerization of the enzyme. The removal of the N-terminal propeptide leads to a structural adjustment of the enzyme and thus promotes enzyme dimerization. IMPORTANCE Vpr-like proteases are widely distributed in bacteria and fungi and are involved in processing lantibiotics, degrading collagen, keratin, and fibrin, and pathogenesis of microbes. The dissection of the roles of individual domains in enzyme maturation and oligomerization is crucial for understanding the action mechanisms of these multidomain proteases. Our results demonstrate that hetero-catalytic maturation of the extracellular Vpr-like protease BTV of Brevibacillus sp. WF146 is more efficient than autocatalytic maturation of the enzyme. Moreover, we found that the C-terminal tandem fibronectin type III domains rather than the PA domain mediate the dimerization of mature BTV, while the N-terminal propeptide inhibits the dimerization of the BTV proform. This study provides new insight into the activation and oligomerization mechanisms of Vpr-like proteases.

Irisin and the fibronectin type III domain-containing family: structure, signaling and role in female reproduction.

Dietary stress such as obesity and short-term changes in energy balance can disrupt ovarian function leading to infertility. Adipose tissue secretes hormones (adipokines), such as leptin and adiponectin, that are known to alter ovarian function. Muscles can also secrete endocrine factors, and one such family of myokines, the eleven Fibronectin type III domain-containing (FNDC) proteins, is emerging as important for energy sensing and homeostasis. In this review, we summarize the known roles the FNDC proteins play in energy homeostasis and explore potential impacts on fertility in females. The most well-known member, FNDC5, is the precursor of the 'exercise hormone', irisin, secreted by both muscle and adipose tissue. The receptors for irisin are integrins, and it has recently been shown to alter steroidogenesis in ovarian granulosa cells although the effects appear to be species or context specific, and irisin may improve uterine and placental function in women and rodent models. Another member, FNDC4, is also cleaved to release a bioactive protein that modulates insulin sensitivity in peripheral tissues and whose receptor, ADGRF5, is expressed in the ovary. As obese women and farm animals in negative energy balance (NEB) both have altered insulin sensitivity, secreted FNDC4 may impact ovarian function. We propose a model in which NEB or dietary imbalance alters plasma irisin and secreted FNDC4 concentrations, which then act on the ovary through their cognate receptors to reduce granulosa cell proliferation and follicle health. Research into these molecules will increase our understanding of energy sensing and fertility and may lead to new approaches to alleviate post-partum infertility. In Brief: Hormones secreted by muscle cells (myokines) are involved in the adaptive response to nutritional and metabolic changes. In this review, we discuss how one family of myokines may alter fertility in response to sudden changes in energy balance.

Allosteric modulation of a human protein kinase with monobodies.

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.

Fibronectin type III domain containing four promotes differentiation of C2C12 through the Wnt/ß-catenin signaling pathway.

Fibronectin type III domain containing 4 (FNDC4) belongs to the fibronectin type III domain containing protein family. FNDC5, which is highly homologous to FNDC4, can promote the differentiation of cardiac cells. We aimed to investigate the role of FNDC4 in the differentiation of C2C12 mouse skeletal muscle cells. Western blotting and immunofluorescence analysis showed that FNDC4 gradually increased with the differentiation of C2C12. Muscle injury repair experiments indicated that FNDC4 may promote the repair of injured muscles. When FNDC4 was either overexpressed or knocked down, the expression of desmin and myogenin myogenic marker molecules followed that of FNDC4, suggesting that FNDC4 can influence the differentiation of C2C12. In addition, immunoprecipitation results showed that FNDC4 can interact with the Wnt/ß-catenin signaling pathway receptor low-density lipoprotein receptor-related protein 6 (LRP6), and that ß-catenin levels in the nucleus decreased after knocking down FNDC4. Exogenous addition of FNDC4 protein could not restore the blocking of differentiation due to inhibition of both Wnt/ß-catenin signal transduction and LRP6 activity via the ß-catenin inhibitor XAV-939. Overall, our findings indicate that FDNC4 can influence the differentiation of C2C12 by activating Wnt/ß-catenin signal transduction.

Fibronectin type III domain-containing 5 in cardiovascular and metabolic diseases: a promising biomarker and therapeutic target.

Cardiovascular and metabolic diseases are the leading causes of death and disability worldwide and impose a tremendous socioeconomic burden on individuals as well as the healthcare system. Fibronectin type III domain-containing 5 (FNDC5) is a widely distributed transmembrane glycoprotein that can be proteolytically cleaved and secreted as irisin to regulate glycolipid metabolism and cardiovascular homeostasis. In this review, we present the current knowledge on the predictive and therapeutic role of FNDC5 in a variety of cardiovascular and metabolic diseases, such as hypertension, atherosclerosis, ischemic heart disease, arrhythmia, metabolic cardiomyopathy, cardiac remodeling, heart failure, diabetes mellitus, and obesity.

DCC/NTN1 complex mutations in patients with congenital hypogonadotropic hypogonadism impair GnRH neuron development.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease characterized by absent puberty and infertility due to GnRH deficiency, and is often associated with anosmia [Kallmann syndrome (KS)]. The genetic etiology of CHH is heterogeneous, and more than 30 genes have been implicated in approximately 50% of patients with CHH. We hypothesized that genes encoding axon-guidance proteins containing fibronectin type-III (FN3) domains (similar to ANOS1, the first gene associated with KS), are mutated in CHH. We performed whole-exome sequencing in a cohort of 133 CHH probands to test this hypothesis, and identified rare sequence variants (RSVs) in genes encoding for the FN3-domain encoding protein deleted in colorectal cancer (DCC) and its ligand Netrin-1 (NTN1). In vitro studies of these RSVs revealed altered intracellular signaling associated with defects in cell morphology, and confirmed five heterozygous DCC mutations in 6 probands-5 of which presented as KS. Two KS probands carry heterozygous mutations in both DCC and NTN1 consistent with oligogenic inheritance. Further, we show that Netrin-1 promotes migration in immortalized GnRH neurons (GN11 cells). This study implicates DCC and NTN1 mutations in the pathophysiology of CHH consistent with the role of these two genes in the ontogeny of GnRH neurons in mice.

Spectroscopic Evidence of the Salt-Induced Conformational Change around the Localized Electric Charges on the Protein Surface of Fibronectin Type III.

The effect of salt on the electrostatic interaction of a protein is an important issue, because addition of salt affects protein stability and association/aggregation. Although adding salt is a generally recognized strategy to improve protein stability, this improvement does not necessarily occur. The lack of an effect upon the addition of salt was previously confirmed for the tenth fibronectin type III domain from human fibronectin (FN3) by thermal stability analysis. However, the detailed molecular mechanism is unknown. In the present study, by employing the negatively charged carboxyl triad on the surface of FN3 as a case study, the molecular mechanism of the inefficient NaCl effect on protein stability was experimentally addressed using spectroscopic methods. Complementary analysis using Raman spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence revealed the three-phase behavior of the salt-protein interaction between NaCl and FN3 over a wide salt concentration range from 100 mM to 4.0 M, suggesting that the Na+-specific binding to the negatively charged carboxyl triad causes a local conformational change around the binding site with an accompanying structural change in the overall protein, which contributes to the protein's structural destabilization. This spectroscopic evidence clarifies the molecular understanding of the inefficiency of salt to improve protein stability. The findings will inform the optimization of formulation conditions.

FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro.

Most species of the genus Bifidobacterium contain the gene cluster PFNA, which is presumably involved in the species-specific communication between bacteria and their hosts. The gene cluster PFNA consists of five genes including fn3, which codes for a protein containing two fibronectin type III domains. Each fibronectin domain contains sites similar to cytokine-binding sites of human receptors. Based on this finding we assumed that this protein would bind specifically to human cytokines in vitro. We cloned a fragment of the fn3 gene (1503 bp; 501 aa) containing two fibronectin domains, from the strain B. longum subsp. longum GT15. After cloning the fragment into the expression vector pET16b and expressing it in E. coli, the protein product was purified to a homogenous state for further analysis. Using the immunoferment method, we tested the purified fragment's ability to bind the following human cytokines: IL-1ß, IL-6, IL-10, TNFα. We developed a sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. We found that the purified protein fragment only binds to TNFα.

Avidity and Cell Uptake of Integrin-Targeting Polypeptide Micelles is Strongly Shape-Dependent.

We describe a genetically encoded micelle for targeted delivery consisting of a diblock polypeptide with segments derived from repetitive protein motifs inspired by Drosophila melanogaster Rec-1 resilin and human tropoelastin with a C-terminal fusion of an integrin-targeting fibronectin type III domain. By systematically varying the weight fraction of the hydrophilic elastin-like polypeptide (ELP) block and molecular weight of the diblock polypeptide, we designed micelles of different morphologies that modulate the binding avidity of the human wild-type 10th fibronectin domain (Fn3) as a function of shape. We show that wormlike micelles that present the Fn3 domain have a 1000-fold greater avidity for the αvß3 receptor compared to the monomer ligand and an avidity that is greater than a clinically relevant antibody that is driven by their multivalency. The amplified avidity of these micelles leads to significantly increased cellular internalization, a feature that may have utility for the intracellular delivery of drugs that are loaded into the core of these micelles.

Urotensin II Induces Mice Skeletal Muscle Atrophy Associated with Enhanced Autophagy and Inhibited Irisin Precursor (Fibronectin Type III Domain Containing 5) Expression in Chronic Renal Failure.

BACKGROUND/AIMS: Skeletal muscle atrophy is one of the main manifestations of protein energy wasting. We hypothesized that urotensin II (UII) can lead to skeletal muscle atrophy through upregulating autophagy and affecting Irisin precursor fibronectin type III domain containing 5 (FNDC5) expressions. METHODS: Three animal models (the sham operation, wild-type C57BL/6 mice with 5/6 nephrectomy, UII receptor (UT) gene knockout (UTKO) mice with 5/6 nephrectomy) were designed. Skeletal muscle weight, cross-sectional area (CSA) along with UII, FNDC5, LC3, and p62 expression were investigated. C2C12 cells were differentiated for up to 4 days into myotubes. These cells were then exposed to different UII concentrations (10-5 to 10-7 M) for 6-12 h and analyzed for the expressions of autophagic markers. These cells were also exposed to the same predetermined UII concentrations for 48-72 h and analyzed for the FNDC5 expression. Myotube diameter was measured. RESULTS: Upregulation of UII expression in skeletal muscle tissue was accompanied by reduced muscle weight and skeletal muscle CSA in the 2 posterior limbs, upregulated autophagy markers expression, and downregulated FNDC5 expression in 5/6 nephrectomy mice. The decrease of skeletal muscle weight, skeletal muscle CSA, downregulation of FNDC5 expression, and the upregulation of autophagy markers were inhibited in UTKO with 5/6 nephrectomy mice. Our in vitrostudy showed that UII could directly decrease myotube diameter, induce autophagy markers upregulation, and inhibit expression of FNDC5. When UII receptor gene was interfered by UT-specific siRNA, UII induced autophagy markers upregulation and FNDC5 downregulation were inhibited. CONCLUSION: We are the first to verify UII induces mice skeletal muscle atrophy associated with enhanced skeletal muscle autophagy and inhibited FNDC5 expression in chronic renal failure.

Extracellular production of recombinant N-glycosylated anti-VEGFR2 monobody in leaky Escherichia coli strain.

OBJECTIVE: To improve the production yield of N-glycosylated anti-VEGFR2 (vascular endothelial growth factor receptor 2) monobody (FN3VEGFR2-Gly) in lpp knockout Escherichia coli cells harboring Campylobacter jejuni N-glycosylation pathway. RESULTS: The leaky CLM37-Δlpp strain efficiently secreted FN3VEGFR2-Gly into culture medium. The extracellular levels of glycosylated FN3VEGFR2-Gly in CLM37-Δlpp culture medium were approximately 11 and 15 times higher compared to those in CLM37 cells via IPTG and auto-induction, respectively. In addition, the highest level of total glycosylated FN3VEGFR2-Gly (70 ± 3.4 mg/L) was found in culture medium via auto-induction. Furthermore, glycosylated FN3VEGFR2-Gly was more stable than unglycosylated FN3VEGFR2-Gly in this expression system, but their bioactivities were relatively similar. CONCLUSIONS: Lpp knockout leaky E. coli strain combined with auto-induction method can enhance the extracellular production of homogenous N-glycosylated FN3VEGFR2-Gly, and facilitate the downstream protein purification. The findings of this study may provide practical implications for the large-scale production and cost-effective harvesting of N-glycosylation proteins.

Design of fibronectin type III domains fused to an elastin-like polypeptide for the osteogenic differentiation of human mesenchymal stem cells.

Extracellular matrix (ECM) including fibronectin (FN) and elastin plays a pivotal role in providing a microenvironment to support tissue regeneration in stem cell therapy. To develop a novel biomimetic ECM for stem cell differentiation, we engineered FN type III 9 and 10 domains fused to elastin-like polypeptides (FN-ELPs). The recombinant FN-ELP fusion protein was expressed in Escherichia coli and purified by inverse transition cycling. Human mesenchymal stem cells (hMSCs) cultured on plates coated with FN-ELP had significantly greater adhesion activity and proliferation than cells grown on non-coated plates. FN-ELP induced the osteogenic differentiation by elevating alkaline phosphatase (ALP) and mineralization activity of hMSCs. Furthermore, the osteogenic marker gene expressions of ALP, collagen type I (Col I), osteopontin (OPN), and transcriptional coactivator with a PDZ-binding motif (TAZ) were increased in hMSCs cultured on plates coated with FN-ELP. We reported a novel biomimetic ECM with potential for bone regeneration that promotes the osteogenic differentiation of hMSCs.

The Structure in Solution of Fibronectin Type III Domain 14 Reveals Its Synergistic Heparin Binding Site.

Fibronectin is a large multidomain protein of the extracellular matrix that harbors two heparin binding sites, Hep-I and Hep-II, which support the heparin-dependent adhesion of melanoma and neuroblastoma cells [Barkalow, F. J. B., and Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818; McCarthy, J. B., et al. (1988) Biochemistry 27, 1380-1388; Drake, S. L., et al. (1993) J. Biol. Chem. 268, 15859-15867]. The stronger heparin/HS binding site on fibronectin, Hep-II, spans fibronectin type III domains 12-14. Previous site-directed mutagenesis, nuclear magnetic resonance (NMR) chemical shift perturbation, and crystallographic structural studies all agree that the main heparin binding site is located on the surface of fibronectin type III domain 13 [Ingham, K. C., et al. (1993) Biochemistry 32, 12548-12553; Sharma, A., et al. (1999) EMBO J. 18, 1468-1479; Sachchidanand, L. O., et al. (2002) J. Biol. Chem. 277, 50629-50635]. However, the "synergy site" for heparin binding located on fibronectin type III domain 14 remained elusive because the actual binding sites could not be identified. Using NMR spectroscopy and isothermal titration calorimetry, we show here that heparin is able to bind to a cationic 'cradle' of fibronectin type III domain 14 formed by the PRARI sequence, which is involved in the integrin α4ß1 interaction [Mould, A. P., and Humphries, M. J. (1991) EMBO J. 10, 4089-4095], and to the flexible loop comprising residues KNNQKSE between the last two ß-strands, D and E, of FN14. Our data reveal that the individual FN14 domain binds to the sulfated sugars Dp8 and Reviparin with affinities similar to those of the individual domain FN13 [Breddin, H. K. (2002) Expert Opin. Pharmacother. 3, 173-182]. It is noteworthy that by introduction of the last ß-strand of FN13 and the linker region between FN type III domains 13 and 14, the perturbation of NMR chemical shifts by heparin is significantly reduced, especially at the PRARI site. This indicates that the Hep-II binding site of fibronectin is mainly located on FN13 and the synergistic binding site on FN14 involves only the KNNQKSE sequence.

Mapping the recognition domains of pneumococcal fibronectin-binding proteins PavA and PavB demonstrates a common pattern of molecular interactions with fibronectin type III repeats.

Colonization of mucosal respiratory surfaces is a prerequisite for the human pathobiont Streptococcus pneumoniae (the pneumococcus) to cause severe invasive infections. The arsenal of pneumococcal adhesins interacts with a multitude of extracellular matrix proteins. A paradigm for pneumococci is their interaction with the adhesive glycoprotein fibronectin, which facilitates bacterial adherence to host cells. Here, we deciphered the molecular interaction between fibronectin and pneumococcal fibronectin-binding proteins (FnBPs) PavA and PavB respectively. We show in adherence and binding studies that the pneumococcal interaction with fibronectin is a non-human specific trait. PavA and PavB target at least 13 out of 15 type III fibronectin domains as demonstrated in ligand overlay assays, surface plasmon resonance studies and SPOT peptide arrays. Strikingly, both pneumococcal FnBPs recognize similar peptides in targeted type III repeats. Structural comparisons revealed that the targeted type III repeat epitopes cluster on the inner strands of both ß-sheets forming the fibronectin domains. Importantly, synthetic peptides of FnIII1 , FnIII5 or FnIII15 bind directly to FnBPs PavA and PavB respectively. In conclusion, our study suggests a common pattern of molecular interactions between pneumococcal FnBPs and fibronectin. The specific epitopes recognized in this study can potentially be tested as antimicrobial targets in further scientific endeavours.