Is There a Functional Role for the Knotted Topology in Protein UCH-L1?

Knotted proteins are present in nature, but there is still an open issue regarding the existence of a universal role for these remarkable structures. To address this question, we used classical molecular dynamics (MD) simulations combined with experiments to investigate the role of the Gordian knot in the catalytic activity of UCH-L1. To create an unknotted form of UCH-L1, we modified its amino acid sequence by truncating several residues from its N-terminus.

Functional and structural investigation of a broadly neutralizing SARS-CoV-2 antibody.

Since its emergence, SARS-CoV-2 has been continuously evolving, hampering the effectiveness of current vaccines against COVID-19. mAbs can be used to treat patients at risk of severe COVID-19. Thus, the development of broadly protective mAbs and an understanding of the underlying protective mechanisms are of great importance.

Structural insight into the ZFAND1-p97 interaction involved in stress granule clearance.

Arsenite-induced stress granule (SG) formation can be cleared by the ubiquitin-proteasome system aided by the ATP-dependent unfoldase p97. ZFAND1 participates in this pathway by recruiting p97 to trigger SG clearance. ZFAND1 contains two An1-type zinc finger domains (ZF1 and ZF2), followed by a ubiquitin-like domain (UBL); but their structures are not experimentally determined.

Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.

Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins.

Tying a true topological protein knot by cyclization.

Knotted proteins are fascinating to biophysicists because of their robust ability to fold into intricately defined three-dimensional structures with complex and topologically knotted arrangements. Exploring the biophysical properties of the knotted proteins is of significant interest, as they could offer enhanced chemical, thermal, and mechanostabilities. A true mathematical knot requires a closed path; in contrast, knotted protein structures have open N- and C-termini.

Altered Protein Dynamics and a More Reactive Catalytic Cysteine in a Neurodegeneration-associated UCHL1 Mutant.

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1, showed higher catalytic activity than wild-type UCHL1 (UCHL1). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood.

Structural insights into the regulation, ligand recognition, and oligomerization of bacterial STING.

The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling pathway plays a critical protective role against viral infections. Metazoan STING undergoes multilayers of regulation to ensure specific signal transduction. However, the mechanisms underlying the regulation of bacterial STING remain unclear.

Structure, dynamics, and stability of the smallest and most complex 7 protein knot.

Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 7 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus.

Folding and functions of knotted proteins.

Topologically knotted proteins have entangled structural elements within their native structures that cannot be disentangled simply by pulling from the N- and C-termini. Systematic surveys have identified different types of knotted protein structures, constituting as much as 1% of the total entries within the Protein Data Bank. Many knotted proteins rely on their knotted structural elements to carry out evolutionarily conserved biological functions.

High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha.

The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we now provide the first experimental evidence for its site-specific glycosylation pattern.

An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments.

Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin.

Application of hybrid biophysical-biochemical methods to unravel the molecular basis for auto-inhibition and activation of protein tyrosine phosphatase TCPTP/PTPN2.

Since the discovery of protein tyrosine phosphorylation as one of the critical post-translational modifications, it has been well known that the activity of protein tyrosine kinases (PTKs) is tightly regulated. On the other hand, protein tyrosine phosphatases (PTPs) are often regarded to act constitutively active, but recently we and others have shown that many PTPs are expressed in an inactive form due to allosteric inhibition by their unique structural features. Furthermore, their cellular activity is highly regulated in a spatiotemporal manner.

Biophysical and structural characterizations of the effects of mutations on the structure-activity relationships of SARS-CoV-2 spike protein.

Mutations on the spike (S) protein of SARS-CoV-2 could induce structural changes that help increase viral transmissibility and enhance resistance to antibody neutralization. Here, we report a robust workflow to prepare recombinant S protein variants and its host receptor angiotensin-convert enzyme 2 (ACE2) by using a mammalian cell expression system. The functional states of the S protein variants are investigated by cryo-electron microscopy (cryo-EM) and negative staining electron microscopy (NSEM) to visualize their molecular structures in response to mutations, receptor binding, antibody binding, and environmental changes.

Elucidation of folding pathways of knotted proteins.

Understanding the mechanisms by which proteins fold and thread into topologically knotted conformations has been challenging because of the apparent complexity associated with the folding and threading events. Nevertheless, many experimental and computational studies have provided insights into the folding pathways of knotted proteins and showed that most of the knotted proteins could spontaneously and reversibly fold into knotted topologies with highly populated intermediates and, at times, through multiple folding pathways. Our laboratory has reported the folding mechanisms of a variety of knotted proteins that have different knot types, ranging from the simplest trefoil 3 knot to the most complex Stevedore's 6 knot.

In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM.

Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, bringing substantial economic losses in the pork industry. The trimeric spike (S) glycoprotein of PEDV is responsible for virus-host recognition, membrane fusion, and is the main target for vaccine development and antigenic analysis.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

Background: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy.

Methods: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants.

Results: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs).

Tumor suppressor BAP1 nuclear import is governed by transportin-1.

Subcellular localization of the deubiquitinating enzyme BAP1 is deterministic for its tumor suppressor activity. While the monoubiquitination of BAP1 by an atypical E2/E3-conjugated enzyme UBE2O and BAP1 auto-deubiquitination are known to regulate its nuclear localization, the molecular mechanism by which BAP1 is imported into the nucleus has remained elusive. Here, we demonstrated that transportin-1 (TNPO1, also known as Karyopherin β2 or Kapβ2) targets an atypical C-terminal proline-tyrosine nuclear localization signal (PY-NLS) motif of BAP1 and serves as the primary nuclear transporter of BAP1 to achieve its nuclear import.

Impacts of Cancer-associated Mutations on the Structure-Activity Relationship of BAP1.

BRAC1 associated protein-1 (BAP1) is a major tumor suppressor involved in many cancers. The deubiquitinase (DUB) activity of BAP1 is essential for its nuclear localization, histone remodeling and proteostasis associated with mitochondrial calcium flux. Loss of the DUB activity due to catalytic mutations within the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) directly contributes to oncogenesis.

Oxidation of catalytic cysteine of human deubiquitinase BAP1 triggers misfolding and aggregation in addition to functional loss.

Deubiquitinating enzymes (DUBs) form a large protease family involved in a myriad of biological and pathological processes, including ROS sensors. ROS-mediated inhibition of their DUB activities is critical for fine-tuning the stress-activated signaling pathways. Here, we demonstrate that the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) is highly sensitive to moderate oxidative stress.

BARD1 is an ATPase activating protein for OLA1.

OLA1 is a P-loop ATPase, implicated in centrosome duplication through the interactions with tumor suppressors BRCA1 and BARD1. Disruption of the interaction of OLA1 with BARD1 results in centrosome amplification. However, the molecular interplay and mechanism of the OLA1-BARD1 complex remain elusive.

The catalytic activity of TCPTP is auto-regulated by its intrinsically disordered tail and activated by Integrin alpha-1.

T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells. TCPTP is a critical component of a variety of key signaling pathways that are directly associated with the formation of cancer and inflammation. Thus, understanding the molecular mechanism of TCPTP activation and regulation is essential for the development of TCPTP therapeutics.

Sialic acid-containing glycolipids mediate binding and viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD.

Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants.

Extensive glycosylation of the spike protein of severe acute respiratory syndrome coronavirus 2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the coronavirus disease of 2019 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain (NTD) that make it refractory to most neutralizing antibodies directed against this domain.