Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids.

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

Metformin inhibits digestive proteases and impairs protein digestion in mice.

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

The strength and cooperativity of KIT ectodomain contacts determine normal ligand-dependent stimulation or oncogenic activation in cancer.

The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells, and interstitial pacemaker cells. Oncogenic KIT mutations play an important "driver" role in gastrointestinal stromal tumors, acute myeloid leukemias, and melanoma, among other cancers. Here we describe the crystal structure of a recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain, rendering KIT tyrosine kinase activity constitutively activated. The structural analysis, together with biochemical and biophysical experiments and detailed analyses of the activities of a variety of oncogenic KIT mutations, reveals that the strength of homotypic contacts and the cooperativity in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regulating normal ligand-dependent or oncogenic RTK activation via a "zipper-like" mechanism for receptor activation.

Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold.

In Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub-cellular localization and recruitment activity. We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Mutations that inhibited dynamic PopZ localization inhibited the recruitment of other factors to cell poles. Mutations in the C-terminal domain also blocked discrete steps in the assembly of higher-order structures. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into filaments, and these structures are important for sub-cellular localization. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter.

Iron acquisition by a commensal bacterium modifies host nutritional immunity during Salmonella infection.

During intestinal inflammation, host nutritional immunity starves microbes of essential micronutrients, such as iron. Pathogens scavenge iron using siderophores, including enterobactin; however, this strategy is counteracted by host protein lipocalin-2, which sequesters iron-laden enterobactin. Although this iron competition occurs in the presence of gut bacteria, the roles of commensals in nutritional immunity involving iron remain unexplored. Here, we report that the gut commensal Bacteroides thetaiotaomicron acquires iron and sustains its resilience in the inflamed gut by utilizing siderophores produced by other bacteria, including Salmonella, via a secreted siderophore-binding lipoprotein XusB. Notably, XusB-bound enterobactin is less accessible to host sequestration by lipocalin-2 but can be "re-acquired" by Salmonella, allowing the pathogen to evade nutritional immunity. Because the host and pathogen have been the focus of studies of nutritional immunity, this work adds commensal iron metabolism as a previously unrecognized mechanism modulating the host-pathogen interactions and nutritional immunity.

Genetic or therapeutic neutralization of ALK1 reduces LDL transcytosis and atherosclerosis in mice.

Low-density lipoprotein (LDL) accumulation in the arterial wall contributes to atherosclerosis initiation and progression1. Activin A receptor-like type 1 (ACVRL1, called activin-like kinase receptor (ALK1)) is a recently identified receptor that mediates LDL entry and transcytosis in endothelial cells (ECs)2,3. However, the role of this pathway in vivo is not yet known. In the present study, we show that genetic deletion of ALK1 in arterial ECs of mice substantially limits LDL accumulation, macrophage infiltration and atherosclerosis without affecting cholesterol or triglyceride levels. Moreover, a selective monoclonal antibody binding ALK1 efficiently blocked LDL transcytosis, but not bone morphogenetic protein-9 (BMP9) signaling, dramatically reducing plaque formation in LDL receptor knockout mice fed a high-fat diet. Thus, our results demonstrate that blocking LDL transcytosis into the endothelium may be a promising therapeutic strategy that targets the initiating event of atherosclerotic cardiovascular disease.

Molecular recognition of leucine-aspartate repeat (LD) motifs by the focal adhesion targeting homology domain of cerebral cavernous malformation 3 (CCM3).

Cerebral cavernous malformation (CCM) is a disease that affects between 0.1 and 0.5% of the human population, with mutations in CCM3 accounting for ~ 15% of the autosomal dominant form of the disease. We recently reported that CCM3 contains an N-terminal dimerization domain (CCM3D) and a C-terminal focal adhesion targeting (FAT) homology domain. Intermolecular protein-protein interactions of CCM3 are mediated by a highly conserved surface on the FAT homology domain and are affected by CCM3 truncations in the human disease. Here we report the crystal structures of CCM3 in complex with three different leucine-aspartate repeat (LD) motifs (LD1, LD2, and LD4) from the scaffolding protein paxillin, at 2.8, 2.7, and 2.5 Å resolution. We show that CCM3 binds LD motifs using the highly conserved hydrophobic patch 1 (HP1) and that this binding is similar to the binding of focal adhesion kinase and Pyk2 FAT domains to paxillin LD motifs. We further show by surface plasmon resonance that CCM3 binds paxillin LD motifs with affinities in the micromolar range, similar to FAK family FAT domains. Finally, we show that endogenous CCM3 and paxillin co-localize in mouse cerebral pericytes. These studies provide a molecular-level framework to investigate the protein-protein interactions of CCM3.

Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition.

c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH--the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control.

Nucleobindin 1 is a calcium-regulated guanine nucleotide dissociation inhibitor of G{alpha}i1.

Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα(i1) and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα(i1) inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα(i)-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα(i1). To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.

Structural characterization of the E2 domain of APL-1, a Caenorhabditis elegans homolog of human amyloid precursor protein, and its heparin binding site.

The amyloid beta-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.

The structural basis of cyclic diguanylate signal transduction by PilZ domains.

The second messenger cyclic diguanylate (c-di-GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c-di-GMP and allosterically modulate effector pathways. We have determined a 1.9 A crystal structure of c-di-GMP bound to VCA0042/PlzD, a PilZ domain-containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain-containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C-terminal PilZ domain plus an N-terminal domain with a similar beta-barrel fold. C-di-GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven-residue long N-terminal loop that undergoes a conformational switch as it wraps around c-di-GMP. This switch brings the PilZ domain into close apposition with the N-terminal domain, forming a new allosteric interaction surface that spans these domains and the c-di-GMP at their interface. The very small size of the N-terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.

Characterization of Protein-Nucleic Acid Complexes by Size-Exclusion Chromatography Coupled with Light Scattering, Absorbance, and Refractive Index Detectors.

Size-exclusion chromatography (SEC) coupled with multiangle light scattering detection (SEC/MALS) enables determination of the molecular weight, oligomeric state, and stoichiometry of protein-nucleic acid complexes in solution. Often such complexes show anomalous behavior on SEC, thus presenting a challenge in determination of molecular weight and stoichiometry based solely on the elution position from SEC. In contrast to analytical ultracentrifugation, the SEC/MALS analysis is not affected by the shape of the complex. Here we describe the use of SEC/MALS for characterization of the stoichiometry of the complex between the reverse transcriptase (RT) domain from group II intron-maturase from Eubacterium rectale and intron RNA, and for monitoring protein dimerization that is driven by interaction between single-stranded DNA upstream of the P1 promoter, known as FUSE and FUSE binding protein-interacting repressor (FIR).

Oligomeric States and Hydrodynamic Properties of Lysyl Oxidase-Like 2.

Lysyl oxidase-like 2 (LOXL2) has emerged as a promising therapeutic target against metastatic/invasive tumors and organ and tissue fibrosis. LOXL2 catalyzes the oxidative deamination of lysine and hydroxylysine residues in extracellular matrix (ECM) proteins to promote crosslinking of these proteins, and thereby plays a major role in ECM remodeling. LOXL2 secretes as 100-kDa full-length protein (fl-LOXL2) and then undergoes proteolytic cleavage of the first two scavenger receptor cysteine-rich (SRCR) domains to yield 60-kDa protein (Δ1-2SRCR-LOXL2). This processing does not affect the amine oxidase activity of LOXL2 in vitro. However, the physiological importance of this cleavage still remains elusive. In this study, we focused on characterization of biophysical properties of fl- and Δ1-2SRCR-LOXL2s (e.g., oligomeric states, molecular weights, and hydrodynamic radii in solution) to gain insight into the structural role of the first two SRCR domains. Our study reveals that fl-LOXL2 exists predominantly as monomer but also dimer to the lesser extent when its concentration is <~1 mM. The hydrodynamic radius (Rh) determined by multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) indicates that fl-LOXL2 is a moderately asymmetric protein. In contrast, Δ1-2SRCR-LOXL2 exists solely as monomer and its Rh is in good agreement with the predicted value. The Rh values calculated from a 3D modeled structure of fl-LOXL2 and the crystal structure of the precursor Δ1-2SRCR-LOXL2 are within a reasonable margin of error of the values determined by SEC-MALS for fl- and Δ1-2SRCR-LOXL2s in mature forms in this study. Based on superimposition of the 3D model and the crystal structure of Δ1-2SRCR-LOXL2 (PDB:5ZE3), we propose a configuration of fl-LOXL2 that explains the difference observed in Rh between fl- and Δ1-2SRCR-LOXL2s in solution.

Quantitative characterization of the interactions among c-myc transcriptional regulators FUSE, FBP, and FIR.

Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR(2)-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR(2)-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.

Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.

Analysis of the cytoplasmic interaction between polycystin-1 and polycystin-2.

Autosomal dominant polycystic kidney disease (ADPKD) arises following mutations of either Pkd1 or Pkd2. The proteins these genes encode, polycystin-1 (PC1) and polycystin-2 (PC2), form a signaling complex using direct intermolecular interactions. Two distinct domains in the C-terminal tail of PC2 have recently been identified, an EF-hand and a coiled-coil domain. Here, we show that the PC2 coiled-coil domain interacts with the C-terminal tail of PC1, but that the PC2 EF-hand domain does not. We measured the K0.5 of the interaction between the C-terminal tails of PC1 and PC2 and showed that the direct interaction of these proteins is abrogated by a PC1 point mutation that was identified in ADPKD patients. Finally, we showed that overexpression of the PC1 C-terminal tail in MDCK cells alters the Ca2+ response, but that overexpression of the PC1 C-terminal tail containing the disease mutation does not. These results allow a more detailed understanding of the mechanism of pathogenic mutations in the cytoplasmic regions of PC1 and PC2.

Rescue of Transgenic Alzheimer's Pathophysiology by Polymeric Cellular Prion Protein Antagonists.

Cellular prion protein (PrPC) binds the scrapie conformation of PrP (PrPSc) and oligomeric ß-amyloid peptide (Aßo) to mediate transmissible spongiform encephalopathy (TSE) and Alzheimer's disease (AD), respectively. We conducted cellular and biochemical screens for compounds blocking PrPC interaction with Aßo. A polymeric degradant of an antibiotic targets Aßo binding sites on PrPC with low nanomolar affinity and prevents Aßo-induced pathophysiology. We then identified a range of negatively charged polymers with specific PrPC affinity in the low to sub-nanomolar range, from both biological (melanin) and synthetic (poly [4-styrenesulfonic acid-co-maleic acid], PSCMA) origin. Association of PSCMA with PrPC prevents Aßo/PrPC-hydrogel formation, blocks Aßo binding to neurons, and abrogates PrPSc production by ScN2a cells. We show that oral PSCMA yields effective brain concentrations and rescues APPswe/PS1ΔE9 transgenic mice from AD-related synapse loss and memory deficits. Thus, an orally active PrPC-directed polymeric agent provides a potential therapeutic approach to address neurodegeneration in AD and TSE.

Reexamination of the role of the amino terminus of SecA in promoting its dimerization and functional state.

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three secA mutants and the corresponding proteins: SecADelta8 lacking residues 2 to 8, SecADelta11 lacking residues 2 to 11, and SecADelta11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecADelta11 or SecADelta11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecADelta11 and SecADelta11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecADelta11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of secA expression and better growth of the SecADelta11/N95 mutant than of the SecADelta11 mutant. Biochemical studies showed that SecADelta11 and SecADelta11/N95 had identical dimerization defects, while SecADelta8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecADelta11 and SecADelta11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.

Human gut Bacteroides capture vitamin B12 via cell surface-exposed lipoproteins.

Human gut Bacteroides use surface-exposed lipoproteins to bind and metabolize complex polysaccharides. Although vitamins and other nutrients are also essential for commensal fitness, much less is known about how commensal bacteria compete with each other or the host for these critical resources. Unlike in Escherichia coli, transport loci for vitamin B12 (cobalamin) and other corrinoids in human gut Bacteroides are replete with conserved genes encoding proteins whose functions are unknown. Here we report that one of these proteins, BtuG, is a surface-exposed lipoprotein that is essential for efficient B12 transport in B. thetaiotaomicron. BtuG binds B12 with femtomolar affinity and can remove B12 from intrinsic factor, a critical B12 transport protein in humans. Our studies suggest that Bacteroides use surface-exposed lipoproteins not only for capturing polysaccharides, but also to acquire key vitamins in the gut.

Oligomeric states of proteins determined by size-exclusion chromatography coupled with light scattering, absorbance, and refractive index detectors.

Size-exclusion chromatography (SEC), coupled with "on-line" static laser light scattering (LS), refractive index (RI), and ultraviolet (UV) detection, provides a universal approach for determination of the molar mass and oligomeric state in solution of native proteins as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Such glycosylated proteins or protein-detergent complexes show anomalous behavior on SEC, thus presenting a challenge to determination of molar mass and oligomeric state in solution. In the SEC-UV/LS/RI approach, SEC serves solely as a fractionation step, while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/LS/RI analysis.