Insight into the autoproteolysis mechanism of the RsgI9 anti-σ factor from Clostridium thermocellum.

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/ß/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.

Internal Fragments Enhance Middle-Down Mass Spectrometry Structural Characterization of Monoclonal Antibodies and Antibody-Drug Conjugates.

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are important large biotherapeutics (∼150 kDa) and high structural complexity that require extensive sequence and structure characterization. Middle-down mass spectrometry (MD-MS) is an emerging technique that sequences and maps subunits larger than those released by trypsinolysis. It avoids potentially introducing artifactual modifications that may occur in bottom-up MS while achieving higher sequence coverage compared to top-down MS. However, returning complete sequence information by MD-MS is still challenging. Here, we show that assigning internal fragments in direct infusion MD-MS of a mAb and an ADC substantially improves their structural characterization. For MD-MS of the reduced NIST mAb, including internal fragments recovers nearly 100% of the sequence by accessing the middle sequence region that is inaccessible by terminal fragments. The identification of important glycosylations can also be improved after the inclusion of internal fragments. For the reduced lysine-linked IgG1-DM1 ADC, we show that considering internal fragments increases the DM1 conjugation sites coverage to 80%, comparable to the reported 83% coverage achieved by peptide mapping on the same ADC (Luo et al. Anal. Chem. 2016, 88, 695-702). This study expands our work on the application of internal fragment assignments in top-down MS of mAbs and ADCs and can be extended to other heterogeneous therapeutic molecules such as multispecifics and fusion proteins for more widespread applications.

Characterization of Molecular Tweezer Binding on α-Synuclein with Native Top-Down Mass Spectrometry and Ion Mobility-Mass Spectrometry Reveals a Mechanism for Aggregation Inhibition.

Parkinson's disease, a neurodegenerative disease that affects 15 million people worldwide, is characterized by deposition of α-synuclein into Lewy Bodies in brain neurons. Although this disease is prevalent worldwide, a therapy or cure has yet to be found. Several small compounds have been reported to disrupt fibril formation. Among these compounds is a molecular tweezer known as CLR01 that targets lysine and arginine residues. This study aims to characterize how CLR01 interacts with various proteoforms of α-synuclein and how the structure of α-synuclein is subsequently altered. Native mass spectrometry (nMS) measurements of α-synuclein/CLR01 complexes reveal that multiple CLR01 molecules can bind to α-synuclein proteoforms such as α-synuclein phosphorylated at Ser-129 and α-synuclein bound with copper and manganese ions. The binding of one CLR01 molecule shifts the ability for α-synuclein to bind other ligands. Electron capture dissociation (ECD) with Fourier transform-ion cyclotron resonance (FT-ICR) top-down (TD) mass spectrometry of α-synuclein/CLR01 complexes pinpoints the locations of the modifications on each proteoform and reveals that CLR01 binds to the N-terminal region of α-synuclein. CLR01 binding compacts the gas-phase structure of α-synuclein, as shown by ion mobility-mass spectrometry (IM-MS). These data suggest that when multiple CLR01 molecules bind, the N-terminus of α-synuclein shifts toward a more compact state. This compaction suggests a mechanism for CLR01 halting the formation of oligomers and fibrils involved in many neurodegenerative diseases.

Structure-based design of nanobodies that inhibit seeding of Alzheimer's patient-extracted tau fibrils.

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.

Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody-Drug Conjugates.

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are two of the most important therapeutic drug classes that require extensive characterization, whereas their large size and structural complexity make them challenging to characterize and demand the use of advanced analytical methods. Top-down mass spectrometry (TD-MS) is an emerging technique that minimizes sample preparation and preserves endogenous post-translational modifications (PTMs); however, TD-MS of large proteins suffers from low fragmentation efficiency, limiting the sequence and structure information that can be obtained. Here, we show that including the assignment of internal fragments in native TD-MS of an intact mAb and an ADC can improve their molecular characterization. For the NIST mAb, internal fragments can access the sequence region constrained by disulfide bonds to increase the TD-MS sequence coverage to over 75%. Important PTM information, including intrachain disulfide connectivity and N-glycosylation sites, can be revealed after including internal fragments. For a heterogeneous lysine-linked ADC, we show that assigning internal fragments improves the identification of drug conjugation sites to achieve a coverage of 58% of all putative conjugation sites. This proof-of-principle study demonstrates the potential value of including internal fragments in native TD-MS of intact mAbs and ADCs, and this analytical strategy can be extended to bottom-up and middle-down MS approaches to achieve even more comprehensive characterization of important therapeutic molecules.

Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis.

Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

Native Top-Down Mass Spectrometry with Collisionally Activated Dissociation Yields Higher-Order Structure Information for Protein Complexes.

Native mass spectrometry (MS) of proteins and protein assemblies reveals size and binding stoichiometry, but elucidating structures to understand their function is more challenging. Native top-down MS (nTDMS), i.e., fragmentation of the gas-phase protein, is conventionally used to derive sequence information, locate post-translational modifications (PTMs), and pinpoint ligand binding sites. nTDMS also endeavors to dissociate covalent bonds in a conformation-sensitive manner, such that information about higher-order structure can be inferred from the fragmentation pattern. However, the activation/dissociation method used can greatly affect the resulting information on protein higher-order structure. Methods such as electron capture/transfer dissociation (ECD and ETD, or ExD) and ultraviolet photodissociation (UVPD) can produce product ions that are sensitive to structural features of protein complexes. For multi-subunit complexes, a long-held belief is that collisionally activated dissociation (CAD) induces unfolding and release of a subunit, and thus is not useful for higher-order structure characterization. Here we show not only that sequence information can be obtained directly from CAD of native protein complexes but that the fragmentation pattern can deliver higher-order structural information about their gas- and solution-phase structures. Moreover, CAD-generated internal fragments (i.e., fragments containing neither N-/C-termini) reveal structural aspects of protein complexes.

Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei.

Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesized that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC-MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant to analyze post-translational modifications (PTMs) without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3-hydroxybutyryl-, crotonyl-, valeryl-, and hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the potential impact of these acyl-protein modifications. The extensive changes in acylation-type, abundance, and modification sites with carbon substrate suggest that protein acylation by RACS may be an important regulator of syntrophy.

Top-down mass spectrometry and assigning internal fragments for determining disulfide bond positions in proteins.

Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS), where only terminal fragments are assigned for disulfide-intact proteins, can access disulfide information, but suffers from low fragmentation efficiency, thereby limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide-intact proteins by 20-60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. The inclusion of internal fragments can extend the sequence information of disulfide-intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into the TD-MS workflow for analyzing disulfide-intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody-drug conjugates.

On the Mechanism of Theta Capillary Nanoelectrospray Ionization for the Formation of Highly Charged Protein Ions Directly from Native Solutions.

Theta capillary nanoelectrospray ionization (θ-nanoESI) can be used to "supercharge" protein ions directly from solution for detection by mass spectrometry (MS). In native top-down MS, the extent of protein charging is low. Given that ions with more charge fragment more readily, increasing charge can enhance the extent of sequence information obtained by top-down MS. For θ-nanoESI, dual-channeled nanoESI emitters are used to mix two solutions in low to sub-µs prior to MS. The mechanism for θ-nanoESI mixing has been reported to primarily occur: (i) in a single shared Taylor cone and in the droplets formed from the Taylor cone or (ii) by the fusion of droplets formed from two separate Taylor cones. Using θ-nanoESI-ion mobility MS, native protein solutions were rapidly mixed with denaturing supercharging solutions to form protein ions in significantly higher charge states and with more elongated structures than those formed by premixing the solutions prior to nanoESI-MS. If θ-nanoESI mixing occurred in the Taylor cone and in the droplets resulting from the single Taylor cone, then the extent of protein charging and unfolding should be comparable to or less than that obtained by premixing solutions. Thus, these data are consistent with mixing occurring via droplet fusion rather than in the Taylor cone prior to ESI droplet formation. These data also suggest that highly charged protein ions can be formed by the near-complete mixing of each solution. The presence of supercharging additives in premixed solutions can suppress volatile electrolyte evaporation, limiting the extent of protein charging compared to when the additive is delivered via one channel of a θ-nanoESI emitter. In θ-nanoESI, the formation of two Taylor cones can presumably result in substantial electrolyte evaporation from the ESI droplets containing native-like proteins prior to droplet fusion, thereby enhancing ion charging.

Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43.

Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer's and Parkinson's diseases1. FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity3. Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43.

The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation.

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades fatty and aromatic acids into acetate, CO2, formate, and H2 that are utilized by methanogens and other hydrogen-consuming microbes. S. aciditrophicus benzoate degradation proceeds by a multistep pathway with many intermediate reactive acyl-coenzyme A species (RACS) that can potentially Nε-acylate lysine residues. Herein, we describe the identification and characterization of acyl-lysine modifications that correspond to RACS in the benzoate degradation pathway. The amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment, enabling a range of acylations located, presumably, on the most extensively acylated proteins throughout the proteome to be studied. Seven types of acyl modifications were identified, six of which correspond directly to RACS that are intermediates in the benzoate degradation pathway including 3-hydroxypimeloylation, a modification first identified in this system. Indeed, benzoate-degrading enzymes are heavily represented among the acylated proteins. A total of 125 sites were identified in 60 proteins. Functional deacylase enzymes are present in the proteome, indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation. Uniquely, Nε-acyl-lysine RACS are highly abundant in these syntrophic bacteria, raising the compelling possibility that post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria. Our results outline candidates for further study of how acylations impact syntrophic consortia.

Towards understanding the formation of internal fragments generated by collisionally activated dissociation for top-down mass spectrometry.

Top-down mass spectrometry (TD-MS) generates fragment ions that returns information on the polypeptide amino acid sequence. In addition to terminal fragments, internal fragments that result from multiple cleavage events can also be formed. Traditionally, internal fragments are largely ignored due to a lack of available software to reliably assign them, mainly caused by a poor understanding of their formation mechanism. To accurately assign internal fragments, their formation process needs to be better understood. Here, we applied a statistical method to compare fragmentation patterns of internal and terminal fragments of peptides and proteins generated by collisionally activated dissociation (CAD). Internal fragments share similar fragmentation propensities with terminal fragments (e.g., enhanced cleavages N-terminal to proline and C-terminal to acidic residues), suggesting that their formation follows conventional CAD pathways. Internal fragments should be generated by subsequent cleavages of terminal fragments and their formation can be explained by the well-known mobile proton model. In addition, internal fragments can be coupled with terminal fragments to form complementary product ions that span the entire protein sequence. These enhance our understanding of internal fragment formation and can help improve sequencing algorithms to accurately assign internal fragments, which will ultimately lead to more efficient and comprehensive TD-MS analysis of proteins and proteoforms.

Internal Fragments Generated from Different Top-Down Mass Spectrometry Fragmentation Methods Extend Protein Sequence Coverage.

Top-down mass spectrometry (TD-MS) of intact proteins results in fragment ions that can be correlated to the protein primary sequence. Fragments generated can either be terminal fragments that contain the N- or C-terminus or internal fragments that contain neither termini. Traditionally in TD-MS experiments, the generation of internal fragments has been avoided because of ambiguity in assigning these fragments. Here, we demonstrate that in TD-MS experiments internal fragments can be formed and assigned in collision-based, electron-based, and photon-based fragmentation methods and are rich with sequence information, allowing for a greater extent of the primary protein sequence to be explained. For the three test proteins cytochrome c, myoglobin, and carbonic anhydrase II, the inclusion of internal fragments in the analysis resulted in approximately 15-20% more sequence coverage, with no less than 85% sequence coverage obtained. Combining terminal fragment and internal fragment assignments results in near complete protein sequence coverage. Hence, by including both terminal and internal fragment assignments in TD-MS analysis, deep protein sequence analysis, allowing for the localization of modification sites more reliably, can be possible.

ClipsMS: An Algorithm for Analyzing Internal Fragments Resulting from Top-Down Mass Spectrometry.

Top-down mass spectrometry (TD-MS) of peptides and proteins results in product ions that can be correlated to polypeptide sequence. Fragments can either be terminal fragments, which contain either the N- or the C-terminus, or internal fragments that contain neither termini. Normally, only terminal fragments are assigned due to the computational difficulties of assigning internal fragments. Here we describe ClipsMS, an algorithm that can assign both terminal and internal fragments generated by top-down MS fragmentation. Further, ClipsMS can be used to locate various modifications on the protein sequence. Using ClipsMS to assign TD-MS generated product ions, we demonstrate that for apo-myoglobin, the inclusion of internal fragments increases the sequence coverage up to 78%. Interestingly, many internal fragments cover complementary regions to the terminal fragments that enhance the information that is extracted from a single top-down mass spectrum. Analysis of oxidized apo-myoglobin using terminal and internal fragment matching by ClipsMS confirmed the locations of oxidation sites on the two methionine residues. Internal fragments can be beneficial for top-down protein fragmentation analysis, and ClipsMS can be a valuable tool for assigning both terminal and internal fragments present in a top-down mass spectrum. Data are available via the MassIVE community resource with the identifiers MSV000086788 and MSV000086789.

Leveraging Immonium Ions for Targeting Acyl-Lysine Modifications in Proteomic Datasets.

Acyl modifications vary greatly in terms of elemental composition and site of protein modification. Developing methods to identify acyl modifications more confidently can help to assess the scope of these modifications in large proteomic datasets. The utility of acyl-lysine immonium ions is analyzed for identifying the modifications in proteomic datasets. It is demonstrated that the cyclized immonium ion is a strong indicator of acyl-lysine presence when its rank or relative abundance compared to other ions within a spectrum is considered. Utilizing a stepped collision energy method in a shotgun experiment highlights the immonium ion. By implementing an analysis that accounted for features within each MS2 spectrum, the method clearly identifies peptides with short chain acyl-lysine modifications from complex lysates. Immonium ions can also be used to validate novel acyl modifications; in this study, the first examples of 3-hydroxylpimelyl-lysine modifications are reported and they are validated using immonium ions. Overall these results solidify the use of the immonium ion as a marker for acyl-lysine modifications in complex proteomic datasets.

Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry.

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. Here, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ∼20-40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ∼50 to ∼99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

Kinetics and Optimization of the Lysine-Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae.

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis.