Identification of Heparin-binding Sites in Proteins by Selective Labeling

Heparan sulfate proteoglycans are key regulators of complex molecular networks due to the interaction of their sugar chains with a large number of partner proteins, which in humans number more than 200 (Ori, A., Wilkinson, M. C., and Fernig, D. G. (2008) The heparanome and regulation of cell function: structures, functions and challenges. Front. Biosci. 13, 4309–4338). We developed a method to selectively label residues involved in heparin binding that matches the requirements for medium/high throughput applications called the “Protect and Label” strategy. This is based on the protection against chemical modification given by heparin/heparan sulfate to the residues located in the heparin-binding site. Thus, analysis of fibroblast growth factor-2 bound to heparin and incubated with N-hydroxysuccinimide acetate showed that lysines involved in the sugar binding are protected against chemical modification. Moreover following release from heparin, the protected lysine side chains may be specifically labeled with N-hydroxysuccinimide biotin. After protein digestion, the biotinylated peptides were readily isolated and identified by MALDI-Q-TOF mass spectrometry. The analysis of labeled peptides obtained from three well characterized heparin-binding proteins with very different heparin-binding sites, fibroblast growth factor-2, platelet factor-4, and pleiotrophin demonstrates the success of this new approach, which thus provides a rapid and reliable procedure to identify heparin-binding sites.Heparan sulfate proteoglycans are a ubiquitous component of the extracellular space of complex organisms (1). They are characterized by their size and their plasticity that derives mainly from the complex and dynamically regulated structure of the glycosaminoglycan (GAG)¹ moiety.They participate in the structural organization of the extracellular space (2, 3) and play an active role in molecular networks driving complex biological phenomena such as development (4–6), inflammation and immune response (7, 8), and disease (9). Heparan sulfate proteoglycans exert their functions by interacting with a vast number of protein partners and so regulating their activity (8, 10).In the last decade, a number of techniques have been used to investigate the interaction between proteins and GAGs (for reviews, see Refs. 11–15). However, despite the identification of more than 200 human heparin-interacting proteins (10, 16), our understanding of the structural features mediating the interaction remains quite poor (10, 13). Some models have been proposed, but they are based on the structural features of only restricted groups or families of heparin-binding proteins (HBPs) (11, 17, 18). Important limitations for modeling of heparin-binding sites (HBSs) are that many interactions are described solely at a qualitative level and that a three-dimensional structure of the sugar-protein complex is available for less than 10% of the annotated interactions (10). The available data derive mainly from x-ray crystallography (e.g. Refs. 19 and 20), NMR spectroscopy (e.g. Ref. 21), and quantitative biophysics with site-directed mutagenesis (e.g. Ref. 22) or HBP-derived synthetic peptides (e.g. Ref. 23). Although providing detailed structural and kinetic information about the interaction, all these methods are limited to the study of single protein-sugar interactions, and they cannot be translated into a high throughput format.In 1989 Chang (24) used a lysine-reactive chromophoric reagent to investigate the HBS of antithrombin III (ATIII). ATIII was modified in the presence or absence of heparin, and the colored peptides generated by tryptic digestion were analyzed by RP-HPLC to obtain important insights into the heparin-ATIII interaction. N-Hydroxysuccinimide (NHS) esters have a strong and selective reactivity toward primary amines, in particular ε-amines on lysines, and their relative stability in neutral/weakly basic aqueous solution makes them compounds of choice for the investigation of HBSs, which are characterized by a high content of basic residues.We developed a rapid and reliable method for the localization of HBSs that can represent an important complement to any structural investigation of heparin-protein interactions because of its low requirement in terms of sample quantity and handling time. The new method, called the “protect and label” strategy, is based on the protection against chemical modification given by heparin/HS to residues located in the HBSs. Thus, an acetyl NHS ester was used to protect residues exposed on the protein surface but not involved in heparin binding, and a second reagent, NHS-biotin, was used to selectively label residues in the HBS. The identification by mass spectrometry of the biotinylated peptides, derived from enzymatic digestion of the HBP, provides a fingerprint of sugar binding on the protein surface.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827.During the last decade, paleontology and taphonomy (the study of decaying organisms over time and the fossilization processes) have begun to overlap with the field of proteomics to shed new light on preserved organic matter in fossilized bones (1–4). These bones represent a time capsule of ancient biomolecules, owing to their natural resistance to post mortem decay arising from a unique combination of mechanical, structural, and chemical properties (4–7).Although bones can be cursorily described as a composite of collagen (protein) and hydroxyapatite (mineral), fossilized bones undergo three distinct diagenesis pathways: (i) chemical deterioration of the organic phase; (ii) chemical deterioration of the mineral phase; and (iii) (micro)biological attack of the composite (6). In addition, the rate of these degradation pathways are affected by temperature, as higher burial temperatures have been shown to accelerate these processes (6, 8). Though relatively unusual, the first of these three pathways results in a slower deterioration process, which is more generally mitigated under (6) specific environmental constraints, such as geochemical stability (stable temperature and acidity) that promote bone mineral preservation. Importantly, slower deterioration results in more preserved biological materials that are more amenable to downstream analytical assays. One example of this is the controversial case of bone and soft-tissue preservation from the Cretaceous/Tertiary boundary (9–22). In light of these and other studies of ancient biomolecules, paleontological models have proposed that organic biomolecules in ancient samples, such as collagen sequences from the 80 million-year-(my)-old Campanian hadrosaur, Brachylophosaurus canadensis (16) or 68-my-old Tyrannosaurus rex, might be protected by the microenvironment within bones. Such spaces are believed to form a protective shelter that is able to reduce the effects of diagenetic events. In addition to collagen, preserved biomolecules include blood proteins, cellular lipids, and DNA (4, 5). While the maximum estimated lifespan of DNA in bones is ∼20,000 years (ky) at 10 °C, bone proteins have an even longer lifespan, making them an exceptional target for analysis to gain relevant insights into fossilized samples (6). Indeed, the survival of collagen, which is considered to be the most abundant bone protein, is estimated to be in the range 340 ky at 20 °C. Similarly, osteocalcin, the second-most abundant bone protein, can persist for ≈45 ky at 20 °C, thus opening an unprecedented analytical window to study extremely old samples (2, 4, 23).Although ancient DNA amplification and sequencing can yield interesting clues and potential artifacts from contaminating agents (7, 24–28), the improved preservation of ancient proteins provides access to a reservoir of otherwise unavailable genetic information for phylogenetic inference (25, 29, 30). In particular, mass spectrometry (MS)-based screening of species-specific collagen peptides has recently been used as a low-cost, rapid alternative to DNA sequencing for taxonomic attribution of morphologically unidentifiable small bone fragments and teeth stemming from diverse archeological contexts (25, 31–33).For over five decades, researchers have presented biochemical evidence for the existence of preserved protein material from ancient bone samples (34–36). One of the first direct measurements was by amino acid analysis, which showed that the compositional profile of ancient samples was consistent with collagens in modern bone samples (37–39). Preservation of organic biomolecules, either from bone, dentin, antlers, or ivory, has been investigated by radiolabeled ¹⁴C fossil dating (40) to provide an avenue of delineating evolutionary divergence from extant species (3, 41, 42). It is also important to note that these parameters primarily depend on ancient bone collagen as the levels remain largely unchanged (a high percentage of collagen is retained, as gleaned by laboratory experiments on bone taphonomy (6)). Additionally, antibody-based immunostaining methods have given indirect evidence of intact peptide amide bonds (43–45) to aid some of the first evidence of protein other than collagen and osteocalcin in ancient mammoth (43) and human specimens (46).In the past, mass spectrometry has been used to obtain MS signals consistent with modern osteocalcin samples (2, 47), and eventually postsource decay peptide fragmentation was used to confirm the identification of osteocalcin in fossil hominids dating back ∼75 ky (48). More recently, modern “bottom-up” proteomic methods were applied to mastodon and T. rex samples (10), complementing immunohistochemistry evidence (13, 17). The results hinted at the potential of identifying peptides from proteolytic digest of well-preserved bone samples. This work also highlighted the importance of minimizing sources of protein contamination and adhering to data publication guidelines (20, 21). In the past few years, a very well-preserved juvenile mammoth referred to as Lyuba was discovered in the Siberian permafrost and analyzed using high-resolution tandem mass spectrometry (29). This study was followed with a report by Wadsworth and Buckley (30) describing the analysis of proteins from 19 bovine bone samples spanning 4 ky to 1.5 my. Both of these groups reported the identification of additional collagen and noncollagen proteins.Recently, a series of large extinct mammal bones were unearthed at a reservoir near Snowmass Village, Colorado, USA (49, 50). The finding was made during a construction project at the Ziegler Reservoir, a fossil site that was originally a lake formed at an elevation of ∼2,705 m during the Bull Lake glaciations ∼140 ky ago (49, 51). The original lake area was ∼5 hectares in size with a total catchment of ∼14 hectares and lacked a direct water flow inlet or outlet. This closed drainage basin established a relatively unique environment that resulted in the exceptional preservation of plant material, insects (52), and vertebrate bones (49). In particular, a cranial specimen from extinct Bison latifrons was unearthed from the Biostratigraphic Zone/Marine Oxygen Isotope Stage (MIS) 5d, which dates back to ∼120 ky (53, 54).Here, we describe the use of paleoproteomics, for the identification of protein remnants with a focus on a particularly unique B. latifrons cranial specimen found at the Ziegler site. We developed a simplified sample processing approach that allows for analysis of low milligram quantities of ancient samples for peptide identification. Our method avoids the extensive demineralization steps of traditional protocols and utilizes an acid labile detergent to allow for efficient extraction and digestion without the need for additional sample cleanup steps. This approach was applied to a specimen from B. latifrons that displayed visual and mechanical properties consistent with the meninges, a fibrous tissue that lines the cranial cavity. Bioinformatics analysis revealed the presence of a recurring glycosylation signature in well-preserved collagens. In particular, the presence of glycosylated hydroxylysine residues was identified as a unique feature of bone fossil collagen, as gleaned through meta-analyses of raw data from previous reports on woolly mammoth (Mammuthus primigenius) and bovine samples (29, 30). The results from these meta-analyses indicate a common, unique feature of collagen that coincides with, and possibly contributes to its preservation.     

Comprehensive Identification of Proteins from MALDI Imaging

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a generic method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. All protein identification data from this study as well as the IMS literature has been deposited into MaTisse, a new publically available database, which we anticipate will become a valuable resource for the IMS community.Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS)¹ is an emerging technique that can be described as a multi-color molecular microscope as it allows visualizing the distribution of many molecules as mass to charge (m/z) signals in parallel in situ (1). Originally described some 15 years ago (2) the method has been successfully adapted to different analyte classes including small molecule drugs (3), metabolites (4), lipids (5), proteins (6), and peptides (7) using e.g. formalin fixed paraffin embedded (FFPE) as well as fresh frozen tissue (8). Because the tissue stays intact in the process, MALDI IMS is compatible with histochemistry (9) as well as immunohistochemistry and thus adds an additional dimension of molecular information to classical microscopy based tissue analysis (10). Imaging of proteins is appealing as it conceptually allows determining the localization and abundance of proteoforms (11) that naturally occur in the tissue under investigation including modifications such as phosphorylation, acetylation, or ubiquitination, protease mediated cleavage or truncation (12). Therefore a proteinous m/z species detected by MALDI IMS can be viewed as an in situ molecular probe of a particular biological process. In turn, m/z abundance patterns that discriminate different physiological or pathological conditions might be used as diagnostic or even prognostic markers (13, 14). In recent years, MALDI IMS of proteins has been successfully applied to different cancer types from the brain (15), breast (16, 17), kidney (18), prostate (19), and skin (20). Furthermore, the technique has been applied in the context of colon inflammation (21), embryonic development (22), Alzheimer''s disease (23), and amyotrophic lateral sclerosis (24). With a few notable exceptions (13, 14, 16–18, 20, 24–30), the identity of the proteins constituting the observed characteristic m/z patters has generally remained elusive. This not only precludes the validation of the putative biomarkers by, for example, immunohistochemistry, but also the elucidation of the biological processes that might underlie the observed phenotype.Here, we introduce a straightforward extraction and identification method for proteins embedded in the MALDI matrix layer that represent the molecular species amenable to MALDI IMS. Using a bottom-up proteomics approach including tryptic digestion and liquid chromatography tandem mass spectrometry (LC-MS/MS), we first created an inventory list of proteins derived from this layer, which we term the MALDI matrix proteome. Although the bottom-up approach breaks the link between the identified proteins and the m/z species detected in MALDI IMS, the list of identified proteins serves as the pool of proteins from which all potential biomarkers are most likely derived. Indeed we detected >90% of all human MALDI IMS biomarkers reported in the literature by analyzing just ten human tissues. In addition, the results demonstrate that the same inventory can be used as a focused database for direct top-down sequencing and identification of proteins extracted from the MALDI matrix layer. The proposed method is generic and can be applied to any MALDI IMS study, which is why we believe that one of the major challenges in identifying MALDI IMS biomarkers has now been overcome. In addition, we provide a list of all proteins and peptides identified in the MALDI matrices and tissues studied here as well as a comprehensive list of m/z species identified in the literature dealing with MALDI imaging of humans and rodents. This information has been compiled in MaTisse (http://www.wzw.tum.de/bioanalytik/matisse), a new publically available and searchable database, which we believe will become a valuable tool for the MALDI imaging community.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

A Straight Path to Circular Proteins

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23).     

ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins

ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction.Energy-dependent proteolysis is responsible for more than 90% of the protein turnover inside the cell (1). This process both removes misfolded and aggregated proteins as part of the response of the cell to stress and controls the concentrations of regulatory proteins (2, 3). In prokaryotes and eukaryotic organelles, energy-dependent proteases fall into five classes as follows: ClpAP, ClpXP, Lon, HslUV (also referred to as ClpYQ), and HflB (also referred to as FtsH). In Archaea, analogous functions are performed by the archaebacterial proteasome, consisting of the proteasome-activating nucleotidase (PAN),³ working with the 20 S proteasome (4); in the cytoplasm and nucleus of eukaryotes, these same functions are performed by the 26 S proteasome (5). These different proteases show little sequence conservation outside the ATP-binding domains, but they share their overall architecture. They all form oligomeric, barrel-shaped complexes composed of one or more rings with the active sites of proteolysis sequestered inside a central degradation chamber (6). Access channels to these sites are narrow, and proteins have to be unfolded to gain entry (6). Regulatory particles belonging to the AAA family of molecular chaperones assemble on either end of the proteolytic chamber and recognize substrates destined for degradation. After recognition, the regulatory particles translocate the substrate through a central channel to the proteolytic chamber and in doing so unravel folded domains within the substrate. Translocation and unfolding are driven by ATP hydrolysis by the regulatory particles, with conformational changes in the protease transmitted to the substrate by conserved residues in the loops lining the channel (7–10).Protein degradation by AAA proteases is tightly regulated. Most proteins are targeted to ClpAP, ClpXP, HslUV, Lon, HflB, and PAN by sequence motifs in their primary structure (11–17). Sometimes adaptor proteins recognize and bind sequence elements in substrates and deliver them to the protease, and other times the protease recognizes sequence elements directly (18, 19). In contrast, proteins are typically targeted to the 26 S proteasome through the covalent attachment of polyubiquitin chains (20). Thus, substrates appear to be selected for degradation based on the presence of specific recognition elements in the protein substrates.However, other mechanisms may also affect the specificity of degradation by prokaryotic proteases. Individual proteases recognize a wide range of targeting signals (11, 16). (For example, Escherichia coli ClpXP recognizes sequences belonging to five distinct classes of consensus sequences (11), and ClpAP, Lon, and FtsH can bind to unstructured regions in proteins with a wide range of amino acid sequences (21–23).) One illustration of the loose specificity in targeting signals is the ability of a mitochondrial presequence to target proteins to the proteases ClpAP (24) and HslUV in vitro (see below). In addition, substrates are commonly acted upon by several different proteases in E. coli. For instance, proteins containing the 11-residue ssrA peptide at their C termini can be recognized by ClpAP, ClpXP, FtsH, Lon, and the archaebacterial proteasome (4, 25–27). Similarly, some substrates of Lon can be degraded by HslUV in vivo (28).It is not clear how degradation remains selective despite the loose specificity of targeting signals. We propose that the intrinsic protein unfolding ability of AAA proteases and the stabilities of substrates against unfolding play a role in determining the fate of cellular proteins. For example, ClpXP releases hard-to-unfold substrates when it encounters them and degrades destabilized titin variants 20-fold faster than wild type titin (29). The membrane-bound AAA protease FtsH has a weak unfolding ability, which allows this protease to act selectively on damaged and unfolded polypeptides (30). Here we find that the relative unfolding abilities of ATP-dependent proteases vary more than 100-fold and that the unfolding abilities of proteases belonging to the same class but originating from different species appear to be conserved. The unfolding abilities also seem to be intrinsic properties of the proteases themselves rather than other cytosolic factors, such as chaperones. Differences in protease unfolding abilities may contribute to substrate selectivity during protein degradation. For example, expression of a protease with a weak unfolding ability during a stress response could allow the selective elimination of unfolded, misfolded, or otherwise aberrant proteins and spare stable proteins from destruction (30).