Protein Structure and Folding
Structure of a dimeric photosystem II complex from a cyanobacterium acclimated to far-red light
Photosystem II (PSII) is the water-splitting enzyme central to oxygenic photosynthesis. To drive water oxidation, light is harvested by accessory pigments, mostly chlorophyll (Chl) a molecules, which absorb visible light (400–700 nm). Some cyanobacteria facultatively acclimate to shaded environments by altering their photosynthetic machinery to additionally absorb far-red light (FRL, 700–800 nm), a process termed far-red light photoacclimation or FaRLiP. During far-red light photoacclimation, FRL-PSII is assembled with FRL-specific isoforms of the subunits PsbA, PsbB, PsbC, PsbD, and PsbH, and some Chl-binding sites contain Chls d or f instead of the usual Chl a.Cryo-EM structure of Shiga toxin 2 in complex with the native ribosomal P-stalk reveals residues involved in the binding interaction
Shiga toxin 2a (Stx2a) is the virulence factor of enterohemorrhagic Escherichia coli. The catalytic A1 subunit of Stx2a (Stx2A1) interacts with the ribosomal P-stalk for loading onto the ribosome and depurination of the sarcin–ricin loop, which halts protein synthesis. Because of the intrinsic flexibility of the P-stalk, a structure of the Stx2a–P-stalk complex is currently unknown. We demonstrated that the native P-stalk pentamer binds to Stx2a with nanomolar affinity, and we employed cryo-EM to determine a structure of the 72 kDa Stx2a complexed with the P-stalk.The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2
Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity.The crystal structure of Vibrio cholerae (6-4) photolyase reveals interactions with cofactors and a DNA-binding region
Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5 Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/β domain, respectively.Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80
Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1.The structure of caseinolytic protease subunit ClpP2 reveals a functional model of the caseinolytic protease system from Chlamydia trachomatis
Chlamydia trachomatis (ct) is the most reported bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Caseinolytic proteases (ClpP) from pathogenic bacteria are attractive antibiotic targets, particularly for bacterial species that form persister colonies with phenotypic resistance against common antibiotics. ClpP functions as a multisubunit proteolytic complex, and bacteria are eradicated when ClpP is disrupted. Although crucial for chlamydial development and the design of agents to treat chlamydia, the structures of ctClpP1 and ctClpP2 have yet to be solved.Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states
PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA–BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA–BV complex.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.
