@article {121922, title = {Contryphan sequence diversity: Messy N-terminus processing, effects on chromatographic behaviour and mass spectrometric fragmentation.}, journal = {J Proteomics}, year = {2023}, month = {2022 Dec 29}, pages = {104805}, abstract = {

Contryphans, peptides containing a single disulfide bond, are found abundantly in cone snail venom. The analysis of a large dataset of available contryphan sequences permits a classification based on the occurrence of proline residues at positions 2 and 5 within the macrocyclic 23-membered disulfide loop. Further sequence diversity is generated by variable proteolytic processing of the contryphan precursor proteins. In the majority of contryphans, presence of Pro at position 2 and a D-residue at position 3 leads to a slow conformational dynamics, manifesting as anomalous chromatographic profiles during LC analysis. LC-MS analysis of diverse contryphans suggests that elution profiles may be used as a rapid diagnostic for the presence of the Pro2-Xxx3 motif. Natural sequences from C.inscriptus and C.frigidus together with synthetic analogs permit the delineation of the features necessary for abnormal chromatographic behaviour. A diagnostic for the presence of Pro at position 5 is obtained by the observation of non-canonical fragment ions, generated by N-C bond cleavage at the dehydroalanine residue formed by disulfide cleavage. Anomalous LC profiles supports Pro at position 2, while non-canonical mass spectral fragments established Pro at position 5, providing a rapid method for contryphan analysis from LC-ESI-MS/MS profiles of crude Conus venom. SIGNIFICANCE: Contryphans are peptides, widely distributed in cone snail venom, which display extensive sequence diversity. Heterogeneity of proteolytic processing of contryphan precursor proteins, together with post-translational modifications contributes to contryphan diversity. Contryphans, identified by a combination of mass spectrometry and transcriptomic analysis, are classified on the basis of sequence features, primarily the number of proline residues within the disulfide loop. Conformational diversity arises in contryphans by cis-trans isomerization of Cys-Pro bonds, resulting in characteristic chromatographic profiles, permitting identification even in crude venom mixtures. Rapid identification of contryphans in cone snail peptide libraries is also facilitated by diagnostic mass spectral fragments arising by non-canonical cleavage of the N-C bond at Cys(6).

}, issn = {1876-7737}, doi = {10.1016/j.jprot.2022.104805}, author = {Vijayasarathy, M and Kumar, Sanjeev and Venkatesha, M A and Balaram, P} } @article {136528, title = {Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.}, journal = {J Pept Sci}, year = {2023}, month = {2023 Nov 27}, pages = {e3554}, abstract = {

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

}, issn = {1099-1387}, doi = {10.1002/psc.3554}, author = {Vijayasarathy, Marimuthu and Kumar, Sanjeev and Das, Rajdeep and Balaram, Padmanabhan} } @article {130549, title = {Decoding the Assembly of Mixed and Branched Heterotypic Ubiquitin Chains.}, journal = {Anal Chem}, volume = {95}, year = {2023}, month = {2023 Jul 04}, pages = {10061-10067}, abstract = {

Ubiquitination is a post-translational modification that regulates cell signaling, immune response, protein processing, molecular trafficking, and DNA repair. Generally, molecular trafficking and DNA repair processes need the attachment of a single ubiquitin on a substrate, known as monoubiquitination. The other functions of ubiquitin require the assembly of polymeric ubiquitin chains on the substrate, known as polyubiquitination. The chains are linked through the lysine residues of ubiquitin, and depending on which lysine is connected, the chains could be heterotypic or homotypic. Heterotypic polyubiquitin chains can be mixed, branched, or combined, generating myriad cellular signals with functions distinct from the homotypic ubiquitin chains. The molecular rules of heterotypic chain assembly are poorly understood due to the lack of techniques to monitor their assembly. New approaches are required to monitor the conjugation site of new ubiquitin molecules on a pre-existing chain. Here, we describe a new method based on isotopic labeling and mass spectrometry to study the assembly of heterotypic chains called isotopically resolved mass spectrometry of peptides (IRMSP). The technique is demonstrated using multiple ubiquitin enzymes and ubiquitin chains as substrates. It causes minimal perturbation to the enzyme/substrate and will be instrumental in studying the assembly of large polymeric ubiquitin chains. Using this technique, it is feasible to monitor how and with what rate branched ubiquitin chains grow in different directions in a single experiment.

}, issn = {1520-6882}, doi = {10.1021/acs.analchem.3c01425}, author = {Singh, Gajendra and Kumar, Sanjeev and Das, Ranabir} } @article {117960, title = {Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme}, journal = {BIOCHEMISTRY}, volume = {61}, year = {2022}, month = {09/2022}, pages = {1988-2006}, abstract = {

Guanosine 5 {\textquoteright}-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5 {\textquoteright}-mono-phosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia channeling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channeling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure, and cross-linking mass spectrometry guided integrative modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being \<= 0.5 s. The crystal structure of the XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.

}, doi = {10.1021/acs.biochem.2c001511988}, author = {Shivakumaraswamy, Santosh and Kumar, Sanjeev and Bellur, Asutosh and Polisetty, Satya Dev and Balaram, Hemalatha} } @article {115053, title = {Mechanistic Investigations on N-C Bond Cleavages in Dibasic Peptides Containing Internal Lys and Arg Residues.}, journal = {J Am Soc Mass Spectrom}, year = {2022}, month = {2022 Jul 26}, abstract = {

The model nonapeptide AAARAAKAG* (* indicates amide) is used to explore N-C bond fragmentation under CID-MS conditions. Neighboring group participation and the effect of positioning of Lys and Arg residues on N-C bond cleavage is established using a library of synthetic peptide analogues. The importance of the Arg residue at position 4 and the to +3 spacing between Arg and Lys residues in determining the formation of the N-C bond cleaved product ions () is demonstrated by a comparative MS study of positional variants in analogue peptides. The effect of shortening of the Lys side chain has been established using ornithine (Orn) and diaminobutyric acid (Dab) analogues. The involvement of the Lys residue in mediating the N-C bond cleavage is further probed using N-dimethyl and isotopically labeled N lysine residues. MS experiments reveal that the ion originates from a doubly charged dehydrated ion [-18]. The mechanism of this unusual fragmentation process has been probed by using position 8 analogues (Gly, Ala, and Aib). A plausible mechanism is proposed for the origin of the ion, which involves C-terminus lactam formation followed by transannular cyclization and dehydration. The results presented in this study highlight the role of reactive side chain functionalities in promoting noncanonical fragmentation pathways.

}, issn = {1879-1123}, doi = {10.1021/jasms.2c00055}, author = {Kumar, Sanjeev and Venkatesha, M Achanna and Balaram, Padmanabhan} } @article {96416, title = {Structural basis for the hyperthermostability of an archaeal enzyme induced by succinimide formation.}, journal = {Biophys J}, year = {2021}, month = {2021 Jul 21}, abstract = {

Stability of proteins from hyperthermophiles (organisms existing under boiling water conditions) enabled by a reduction of conformational flexibility is realized through various mechanisms. A succinimide (SNN) arising from the post-translational cyclization of the side chains of aspartyl/asparaginyl residues with the backbone amide -NH of the succeeding residue would restrain the torsion angle Ψ and can serve as a new route for hyperthermostability. However, such a succinimide is typically prone to hydrolysis, transforming to either an aspartyl or β-isoaspartyl residue. Here, we present the crystal structure of Methanocaldococcus jannaschii glutamine amidotransferase (MjGATase) and using enhanced sampling molecular dynamics simulations, address the mechanism of its increased thermostability, up to 100 C, imparted by an unexpectedly stable succinimidyl residue (SNN) at position 109. The stability of SNN109 to hydrolysis is seen to arise from its electrostatic shielding by the side chain carboxylate group of its succeeding residue Asp110 as well as through n{\textrightarrow}π interactions between SNN109 and its preceding residue Glu108, both of which prevent water access to SNN. The stable succinimidyl residue induces the formation of an α-turn structure involving 13-atom hydrogen bonding which locks the local conformation, reducing protein flexibility. The destabilization of the protein upon replacement of SNN with a Φ-restricted prolyl residue highlights the specificity of the succinimidyl residue in imparting hyperthermostability to the enzyme. The conservation of succinimide-forming tripeptide sequence (E(N/D)(E/D)) in several archaeal GATases strongly suggests an adaptation of this otherwise detrimental post-translational modification as a harbinger of thermostability.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2021.07.014}, author = {Dongre, Aparna Vilas and Das, Sudip and Bellur, Asutosh and Kumar, Sanjeev and Chandrashekarmath, Anusha and Karmakar, Tarak and Balaram, Padmanabhan and Balasubramanian, Sundaram and Balaram, Hemalatha} } @article {73044, title = {Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins.}, journal = {Biochim Biophys Acta Proteins Proteom}, year = {2020}, month = {2020 Feb 10}, pages = {140391}, abstract = {

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (* indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

}, issn = {1878-1454}, doi = {10.1016/j.bbapap.2020.140391}, author = {Kumar, Sanjeev and Vijayasarathy, M and Venkatesha, M A and Sunita, P and Balaram, P} } @article {77446, title = {Mechanistic Insights into an Unusual Side-Chain-Mediated N-C Bond Cleavage under Collision-Induced Dissociation Conditions in the Disulfide-Containing Peptide Conopressin.}, journal = {J Am Soc Mass Spectrom}, volume = {31}, year = {2020}, month = {2020 May 06}, pages = {1083-1092}, abstract = {

Conopressin, a nonapeptide disulfide CFIRNCPKG amide present in cone snail venom, undergoes a facile cleavage at the Cys6-Pro7 peptide bond to yield a disulfide bridged ion. Analysis of the mass spectral fragmentation pattern reveals the presence of a major fragment ion, which is unambiguously assigned as the tripeptide sequence IRN amide. The sequence dependence of this unusual fragmentation process has been investigated by comparing it with the fragmentation patterns of related peptides, oxytocin (CYIQNCPLG amide), Lys-vasopressin (CYFQNCPKG amide), and a series of synthetic analogues. The results establish the role of the Arg4 residue in facilitating the unusual N-C bond cleavage at Cys6. Structures are proposed for a modified disulfide bridged fragment containing the Cys1 and Cys6 residues. Gas-phase molecular dynamics simulations provide evidence for the occurrence of conformational states that permit close approach of the Arg4 side chain to the Cys6 C methylene protons.

}, issn = {1879-1123}, doi = {10.1021/jasms.0c00023}, author = {Kumar, Sanjeev and Venkatesha, M Achanna and Lall, Sahil and Prakash, Sunita and Balaram, Padmanabhan} }