Protein Structure and Folding
The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions
Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions.Determinants of receptor tyrosine phosphatase homophilic adhesion: Structural comparison of PTPRK and PTPRM extracellular domains
Type IIB receptor protein tyrosine phosphatases are cell surface transmembrane proteins that engage in cell adhesion via their extracellular domains (ECDs) and cell signaling via their cytoplasmic phosphatase domains. The ECDs of type IIB receptor protein tyrosine phosphatases form stable, homophilic, and trans interactions between adjacent cell membranes. Previous work has demonstrated how one family member, PTPRM, forms head-to-tail homodimers. However, as the interface was composed of residues conserved across the family, the determinants of homophilic specificity remain unknown.Exploration of the cysteine reactivity of human inducible Hsp70 and cognate Hsc70
Hsp70s are multifunctional proteins and serve as the central hub of the protein quality control network. Hsp70s are also related to a number of diseases and have been established as drug targets. Human HspA1A (hHsp70) and HspA8 (hHsc70) are the major cytosolic Hsp70s, and they have both overlapping and distinct functions. hHsp70 contains five cysteine residues, and hHsc70 contains four cysteine residues. Previous studies have shown these cysteine residues can undergo different cysteine modifications such as oxidation or reaction with electrophiles to regulate their function, and hHsp70 and hHsc70 have different cysteine reactivity.Structures of a constitutively active mutant of human IDH3 reveal new insights into the mechanisms of allosteric activation and the catalytic reaction
Human NAD-dependent isocitrate dehydrogenase or IDH3 (HsIDH3) catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the tricarboxylic acid cycle. It consists of three types of subunits (α, β, and γ) and exists and functions as the (αβαγ)2 heterooctamer. HsIDH3 is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. Our previous studies have revealed the molecular basis for the activity and regulation of the αβ and αγ heterodimers.Dimers of D76N-β2-microglobulin display potent antiamyloid aggregation activity
Self-association of WT β2-microglobulin (WT-β2m) into amyloid fibrils is associated with the disorder dialysis related amyloidosis. In the familial variant D76N-β2m, the single amino acid substitution enhances the aggregation propensity of the protein dramatically and gives rise to a disorder that is independent of renal dysfunction. Numerous biophysical and structural studies on WT- and D76N-β2m have been performed in order to better understand the structure and dynamics of the native proteins and their different potentials to aggregate into amyloid.Macromolecular crowding amplifies allosteric regulation of T-cell protein tyrosine phosphatase
T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of T-cell receptor and oncogenic receptor tyrosine kinase signaling and implicated in cancer and autoimmune disease. TC-PTP activity is modulated by an intrinsically disordered C-terminal region (IDR) and suppressed in cells under basal conditions. In vitro structural studies have shown that the dynamic reorganization of IDR around the catalytic domain, driven by electrostatic interactions, can lead to TC-PTP activity inhibition; however, the process has not been studied in cells.Analysis of Plasmodium falciparum myosin B ATPase activity and structure in complex with the calmodulin-like domain of its light chain MLC-B
Myosin B (MyoB) is a class 14 myosin expressed in all invasive stages of the malaria parasite, Plasmodium falciparum. It is not associated with the glideosome complex that drives motility and invasion of host cells. During red blood cell invasion, MyoB remains at the apical tip of the merozoite but is no longer observed once invasion is completed. MyoB is not essential for parasite survival, but when it is knocked out, merozoites are delayed in the initial stages of red blood cell invasion, giving rise to a growth defect that correlates with reduced invasion success.Structural studies of human fission protein FIS1 reveal a dynamic region important for GTPase DRP1 recruitment and mitochondrial fission
Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal “arm” of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1.Crystal structure of the collagen prolyl 4-hydroxylase (C-P4H) catalytic domain complexed with PDI: Toward a model of the C-P4H α2β2 tetramer
Collagen prolyl 4-hydroxylases (C-P4H) are α2β2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the β-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the β/PDI subunit, are unknown.Structural studies of SALL family protein zinc finger cluster domains in complex with DNA reveal preferential binding to an AATA tetranucleotide motif
The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different dsDNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 recognize an AATA tetranucleotide.Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition
Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C.The extracellular domain of site-2-metalloprotease RseP is important for sensitivity to bacteriocin EntK1
Enterocin K1 (EntK1), a bacteriocin that is highly potent against vancomycin-resistant enterococci, depends on binding to an intramembrane protease of the site-2 protease family, RseP, for its antimicrobial activity. RseP is highly conserved in both EntK1-sensitive and EntK1-insensitive bacteria, and the molecular mechanisms underlying the interaction between RseP and EntK1 and bacteriocin sensitivity are unknown. Here, we describe a mutational study of RseP from EntK1-sensitive Enterococcus faecium to identify regions of RseP involved in bacteriocin binding and activity.Three-dimensional structure of a mycobacterial oligoribonuclease reveals a unique C-terminal tail that stabilizes the homodimer
Oligoribonucleases (Orns) are highly conserved DnaQ-fold 3′-5′ exoribonucleases that have been found to carry out the last step of cyclic-di-GMP (c-di-GMP) degradation, that is, pGpG to GMP in several bacteria. Removal of pGpG is critical for c-di-GMP homeostasis, as excess uncleaved pGpG can have feedback inhibition on phosphodiesterases, thereby perturbing cellular signaling pathways regulated by c-di-GMP. Perturbation of c-di-GMP levels not only affects survival under hypoxic, reductive stress, or nutrient-limiting conditions but also affects pathogenicity in infection models as well as antibiotic response in mycobacteria.Anti-CRISPR protein AcrIF4 inhibits the type I-F CRISPR-Cas surveillance complex by blocking nuclease recruitment and DNA cleavage
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides prokaryotes with protection against mobile genetic elements such as phages. In turn, phages deploy anti-CRISPR (Acr) proteins to evade this immunity. AcrIF4, an Acr targeting the type I-F CRISPR-Cas system, has been reported to bind the crRNA-guided surveillance (Csy) complex. However, it remains controversial whether AcrIF4 inhibits target DNA binding to the Csy complex. Here, we present structural and mechanistic studies into AcrIF4, exploring its unique anti-CRISPR mechanism.Anionic lipid vesicles have differential effects on the aggregation of early onset-associated α-synuclein missense mutants
α-synuclein (αS) is the key component of synucleinopathies such as Parkinson’s disease (PD), dementia with Lewy bodies, and multiple system atrophy. αS was first linked to PD through the identification of point mutations in the SNCA gene, causing single amino acid substitutions within αS and familial autosomal dominant forms of PD that profoundly accelerated disease onset by up to several decades. At least eight single-point mutations linked to familial PD (A30G/P, E46K, H50Q, G51D, and A53T/E/V) are located in proximity of the region preceding the non-β amyloid component (preNAC) region, strongly implicating its pathogenic role in αS-mediated cytotoxicity.Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10
Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood.Molecular characterization of the type VI secretion system effector Tlde1a reveals a structurally altered LD-transpeptidase fold
The type VI secretion system (T6SS) is a molecular machine that Gram-negative bacteria have adapted for multiple functions, including interbacterial competition. Bacteria use the T6SS to deliver protein effectors into adjacent cells to kill rivals and establish niche dominance. Central to T6SS-mediated bacterial competition is an arms race to acquire diverse effectors to attack and neutralize target cells. The peptidoglycan has a central role in bacterial cell physiology, and effectors that biochemically modify peptidoglycan structure effectively induce cell death.Regulation of factor V by the anticoagulant protease activated protein C: Influence of the B-domain and TFPIα
Activated protein C (APC) is an important anticoagulant protein that regulates thrombin generation through inactivation of factor V (FV) and activated factor V (FVa). The rate of APC inactivation of FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg306, Arg506, and Arg679). The molecular basis for FV resistance to APC is unknown. Further, there is no information about how FV-short, a physiologically relevant isoform of FV with a shortened B-domain, is regulated by APC.Biochemical and structural basis for the pharmacological inhibition of nuclear hormone receptor PPARγ by inverse agonists
Recent studies have reported that the peroxisome proliferator–activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines.Plant vernalization proteins contain unusual PHD superdomains without histone H3 binding activity
PHD fingers are modular domains in chromatin-associated proteins that decode the methylation status of histone H3 tails. A PHD finger signature is found in plant vernalization (VEL) proteins, which function as accessory factors of the Polycomb system to control flowering in Arabidopsis through an epigenetic silencing mechanism. It has been proposed that VEL PHD fingers bind to methylated histone H3 tails to facilitate association of the Polycomb silencing machinery with target genes. Here, we use structural analysis by X-ray crystallography to show that the VEL PHD finger forms the central module of a larger compact tripartite superdomain that also contains a zinc finger and a four-helix bundle.Natural polyphenols convert proteins into histone-binding ligands
Antioxidants are sensitive to oxidation and are immediately converted into their oxidized forms that can react with proteins. We have recently found that proteins incubated with oxidized vitamin C (dehydroascorbate) gain a new function as a histone-binding ligand. This finding led us to predict that antioxidants, through conversion to their oxidized forms, may generally have similar functions. In the present study, we identified several natural polyphenols as a source of histone ligands and characterized the mechanism for the interaction of protein-bound polyphenols with histone.The staphylococcal inhibitory protein SPIN binds to human myeloperoxidase with picomolar affinity but only dampens halide oxidation
The heme enzyme myeloperoxidase (MPO) is one of the key players in the neutrophil-mediated killing of invading pathogens as part of the innate immune system. MPO generates antimicrobial oxidants, which indiscriminately and effectively kill phagocytosed pathogens. Staphylococcus aureus, however, is able to escape this fate, in part by secreting a small protein called SPIN (Staphylococcal Peroxidase Inhibitor), which specifically targets and inhibits MPO in a structurally complex manner. Here, we present the first crystal structures of the complex of SPIN-aureus and a truncated version (SPIN-truncated) with mature dimeric leukocyte MPO.Structural and functional studies of legumain–mycocypin complexes revealed a competitive, exosite-regulated mode of interaction
Under pathophysiologic conditions such as Alzheimer’s disease and cancer, the endolysosomal cysteine protease legumain was found to translocate to the cytosol, the nucleus, and the extracellular space. These noncanonical localizations demand for a tight regulation of legumain activity, which is in part conferred by protein inhibitors. While there is a significant body of knowledge on the interaction of human legumain with endogenous cystatins, only little is known on its regulation by fungal mycocypins.New insights into P2X7 receptor regulation: Ca2+-calmodulin and GDP bind to the soluble P2X7 ballast domain
P2X7 receptors are nonselective cation channels that are activated by extracellular ATP and play important roles in inflammation. They differ from other P2X family members by a large intracellular C-terminus that mediates diverse signaling processes that are little understood. A recent cryo-EM study revealed that the C-terminus of the P2X7 receptor forms a unique cytoplasmic ballast domain that possesses a GDP-binding site as well as a dinuclear Zn2+ site. However, the molecular basis for the regulatory function of the ballast domain as well as the interplay between the various ligands remain unclear.High-resolution structures of a siderophore-producing cyclization domain from Yersinia pestis offer a refined proposal of substrate binding
Nonribosomal peptide synthetase heterocyclization (Cy) domains generate biologically important oxazoline/thiazoline groups found in natural products, including pharmaceuticals and virulence factors such as some siderophores. Cy domains catalyze consecutive condensation and cyclodehydration reactions, although the mechanism is unknown. To better understand Cy domain catalysis, here we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis.
