MrParse: Finding homologues in the PDB and the EBI AlphaFold database for Molecular Replacement and more

Crystallographers have an array of search model options for structure solution by Molecular Replacement (MR). Well-established options of homologous experimental structures and regular secondary structure elements or motifs are increasingly supplemented by computational modelling. Such modelling may be carried out locally or use pre-calculated predictions retrieved from databases such as the EBI AlphaFold database. MrParse is a new pipeline to help streamline the decision process in MR by consolidating bioinformatic predictions in one place. When reflection data are provided, MrParse can rank any homologues found using eLLG which indicates the likelihood that a given search model will work in MR. In-built displays of predicted secondary structure, coiled-coil and transmembrane regions further inform the choice of MR protocol. MrParse can also identify and rank homologues in the EBI AlphaFold database, a function that will also interest other structural biologists and bioinformaticians.

Folding of Truncated Granulin Peptides

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a “mini-granulin” structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.

Structural details of amyloid beta oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy

ABSTRACTHuman PrP (huPrP) is a high-affinity receptor for oligomeric Aβ. Synthetic oligomeric Aβ species are known to be heterogeneous, dynamic and transient, rendering their structural investigation particularly challenging. Here, we used huPrP to preserve Aβ oligomers by co-precipitating them into large hetero-assemblies to investigate the conformation of Aβ(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the last two α-helices. For Aβ(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal a substantial β-strand content. Importantly, not all Aβ(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic Aβ fibrils suggests that the Aβ oligomer preparation represents a heterogeneous mixture of β-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of Aβ to adopt variable β-structure conformers.

A bottom-up approach for the de novo design of functional proteins

De novo protein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to build de novo proteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. The de novo proteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a “CPDCP-trunk” and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the “legumain-binding loop” of CY1 inhibit crayfish astacin following the “raised-elephant-trunk mechanism” recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and β only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Structure of mammalian plasma fetuin-B and its mechanism of selective metallopeptidase inhibition

Mammalian fetuin-A and fetuin-B are abundant serum proteins with pleiotropic functions. Fetuin-B is a highly selective and potent inhibitor of metallopeptidases (MPs) of the astacin family, which includes ovastacin in mammals. By inhibiting ovastacin, fetuin-B is essential for female fertility. The crystal structure of fetuin-B was determined unbound and in complex with archetypal astacin, and it was found that the inhibitor has tandem cystatin-type modules (CY1 and CY2). They are connected by an exposed linker with a rigid, disulfide-linked `CPDCP-trunk', and are followed by a C-terminal region (CTR) with little regular secondary structure. The CPDCP-trunk and a hairpin of CY2 form a bipartite wedge, which slots into the active-site cleft of the MP. These elements occupy the nonprimed and primed sides of the cleft, respectively, but spare the specificity pocket so that the inhibitor is not cleaved. The aspartate in the trunk blocks the catalytic zinc of astacin, while the CY2 hairpin binds through a QWVXGP motif. The CY1 module assists in structural integrity and the CTR is not involved in inhibition, as verified by in vitro studies using a cohort of mutants and variants. Overall, the inhibition conforms to a novel `raised-elephant-trunk' mechanism for MPs, which is reminiscent of single-domain cystatins that target cysteine peptidases. Over 200 sequences from vertebrates have been annotated as fetuin-B, underpinning its ubiquity and physiological relevance; accordingly, sequences with conserved CPDCP- and QWVXGP-derived motifs have been found from mammals to cartilaginous fishes. Thus, the raised-elephant-trunk mechanism is likely to be generally valid for the inhibition of astacins by orthologs of fetuin-B.

Peptide Secondary Structure Prediction using Evolutionary Information

ABSTRACTBACKGROUNDIn the past, large numbers of methods have been developed for predicting secondary structure of proteins. Best of author’s knowledge no method has been specifically developed for predicting secondary structure of peptides. We analyzed secondary structure of peptides and proteins; it was observed that same peptide in protein adopt different secondary structures. Considering the wide application of peptides in therapeutic market, we made attempt to develop a method called PEP2D for predicting secondary structure of peptides.RESULTSIn this study, 3107 unique peptides have been used to train, test and evaluate peptide secondary structure prediction models. It was observed that regular secondary structure content (e.g., helix, beta-sheet) increased with length of peptides. Firstly, models based on various machine-learning techniques have been developed using binary profile of peptides and achieved maximum overall accuracy (Q3) 79.5%. The performance of models further improved from 79.5% to 83.5% using evolutionary information in the form of PSSM profile. We also evaluate performance of protein secondary structure prediction method PSIPRED on our dataset and achieved maximum accuracy 76.9%; particularly poor (Q3 71.4%) for small peptides having length less than 10 residues. Overall, PEP2D has better prediction of beta-sheets (Q3 74%) and coil region (Q3 87%) of peptides as compare to PSIPRED (Q3 54.4% for beta-sheet and Q3 77.9% for coil). We also measure performance of PSIPRED and PEP2D in terms of segment overlap (SOV); achieved 69.3 and 76.7 respectively.CONCLUSIONOur observations indicate that there is a need of developing separate method for predicting secondary structure of peptides. It was also observed that models based on PSSM profile perform poor on small peptides in comparison to long peptides. Based on our study, we developed method for predicting secondary structure of peptides. In order to provide service to user, a webserver/standalone has been developed (https://webs.iiitd.edu.in/raghava/pep2d/).

Pi-Pi contacts are an overlooked protein feature relevant to phase separation

Protein phase separation is implicated in formation of membraneless organelles, signaling puncta and the nuclear pore. Multivalent interactions of modular binding domains and their target motifs can drive phase separation. However, forces promoting the more common phase separation of intrinsically disordered regions are less understood, with suggested roles for multivalent cation-pi, pi-pi, and charge interactions and the hydrophobic effect. Known phase-separating proteins are enriched in pi-orbital containing residues and thus we analyzed pi-interactions in folded proteins. We found that pi-pi interactions involving non-aromatic groups are widespread, underestimated by force-fields used in structure calculations and correlated with solvation and lack of regular secondary structure, properties associated with disordered regions. We present a phase separation predictive algorithm based on pi interaction frequency, highlighting proteins involved in biomaterials and RNA processing.

Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei

ABSTRACTN-linked glycosylation modulates and diversifies the structures and functions of the eukaryotic proteome through both intrinsic and extrinsic effects on proteins. We investigated the significance of the three N-linked glycans on the catalytic domain of cellobiohydrolase I (CBH1) from the filamentous fungusTrichoderma reeseiin its secretion and activity. While the removal of one or two N-glycosylation sites hardly affected the extracellular secretion of CBH1, eliminating all of the glycosylation sites did induce expression of the unfolded protein response (UPR) target genes, and secretion of this CBH1 variant was severely compromised in a calnexin gene deletion strain. Further characterization of the purified CBH1 variants showed that, compared to Asn270, the thermal reactivity of CBH1 was significantly decreased by removal of either Asn45 or Asn384 glycosylation site during the catalyzed hydrolysis of soluble substrate. Combinatorial loss of these two N-linked glycans further exacerbated the temperature-dependent inactivation. In contrast, this thermal labile property was less severe when hydrolyzing insoluble cellulose. Analysis of the structural integrity of CBH1 variants revealed that removal of N-glycosylation at Asn384 had a more pronounced effect on the integrity of regular secondary structure compared to the loss of Asn45 or Asn270. These data implicate differential roles of N-glycosylation modifications in contributing to the stability of specific functional regions of CBH1 and highlight the potential of improving the thermostability of CBH1 by tuning proper interactions between glycans and functional residues.

Phytolacca americana lectin (Pa-2; pokeweed mitogen): an intrinsically unordered protein and its conversion into partial order at low pH

This is the first report of its kind that well demonstrates that a lectin from Phytolacca americana [Pa-2 (P. americana lectin-2)] can also be intrinsically unordered, based on the results obtained by CD, tryptophan fluorescence, ANS (8-anilinonaphthalene-1-sulfonic acid) binding, acrylamide quenching, DLS (dynamic light scattering) and its amino acid composition database analyses. Pa-2 is an acidic monomeric lectin and acquires random coil conformation at neutral pH without any regular secondary structure. As confirmed by different spectroscopic techniques, on lowering the pH, some secondary structures, predominantly α-helices, are detected by far-UV CD that adopt a marginally stable partially folded collapsed conformation possessing the characteristics of a premolten globule state. It is in accordance with coil–helix transition that is commonly observed when these intrinsically unordered proteins interact with their partner molecules in vivo.