29 research outputs found
Protein target highlights in CASP15: Analysis of models by structure providers
We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology
Bestun á kristöllunarskilyrðum Vibrio alkalísks fosfatasa til greingar á bindingu í hvarfstöð
Vibrio alkaline phosphatase (VAP), a cold-adapted variant of the alkaline phosphatase
enzyme superfamily, was here under investigation. Unexpectedly, the enzyme has been
shown to be inhibited by various small organic molecules. Furthermore, our group
recently showed that anions, and chloride ions in particular, have a drastic effect on the
catalytic properties of the enzyme. In order to assess the molecular mechanisms
involved in the binding of these inhibitors and chloride, X-ray crystallography was
performed. This project in turn had two goals, one of optimizing crystallization
conditions producing either apo enzyme crystals to use in crystal soaking or
ligand-protein crystals, via co-crystallization. The other goal was analyzing the active
site binding of chloride by co-crystallizing the enzyme with sodium chloride.
Presented here within are three crystal structures of VAP with bound chloride ions,
obtained from crystals grown under fully optimized conditions. The crystal structures
reveal novel structural features both at the dimer interface of the protein, and most
prominently, in the active site. Chloride ion active site binding is coordinated in part
by the Zn2+ in the M1 metal binding site and Arg129. This suggests that chloride
ions might increase the catalysis rate by competing with the product for active site
binding, promoting product release, as had been previously hypothesized. Furthermore,
the crystal structures seem to reveal asymmetrical binding of anions in the two active
sites of the enzyme. If true, this would be the first asymmetrical AP crystal structure
and would give structural evidence for the proposed half-of-sites reactivity.Hinn kuldaaðlagaði alkalíski fosfatasi úr bakteríunni Vibrio splendidus (VAP) var hér til
rannsóknar. Sýnt hefur verið fram á að ensímið er hindrað á óvæntan hátt af lífrænum
smásameindum. Einnig hafa anjónir, og þá sérstaklega klórjónir, gríðarleg áhrif á
hvötunargetu ensímsins. Til að greina hindrun og hvernig klórjónir bindast á ensímið
var notast við röntgenkristallagreiningu. Markmið verkefnisins var tvíþátta; í fyrsta lagi
að besta kristöllunarskilyrði sem gæfu kristalla með tóma hvarfstöð til að nota í
kristalbleytitilraunum og í öðru lagi að samkristalla með klórjónum eða hindrum.
Í þessu verkefni tókst að greina þrjár nýjar VAP kristalbyggingar með bundnum
klórjónum, sem fengust úr fullbestuðum kristöllunarskilyrðum. Kristalbyggingarnar
sýndu nýstárlega byggingarlega eiginleika próteinsins, og þá sérstaklega á mótum
einliðanna tveggja og í hvarfstöðvum ensímsins. Klórjónir voru bundar í hvarfstöð og
bindingunni er miðlað af Zn2+ jóninni í M1 málmbindisetinu og Arg129. Niðurstöðurnar
renna stöðum undir áður framsetta tilgátu um að klórjónir auki hvötunargetu með
samkeppni við myndefnið um bindingu í hvarfstöð. Kristalbyggingarnar virðast enn
fremur sýna ósamhverfa bindingu anjóna í hvarfstöðvunum tveimur. Ef ósamhverfan er
í raun til staðar eru þetta fyrstu ósamhverfu kristalbyggingar alkalísks fosfatasa, og
renna þar með styrkum stoðum undir kenningar um hálfsetahvötun þessara ensíma
Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase
Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0–1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small-angle X-ray scattering showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability
X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine
Background - Para-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs. Methods - Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-active Vibrio AP (VAP) was performed and the structure solved. Results - Inhibition of VAP fitted a non-competitive kinetic model (Km unchanged, Vmax reduced) with IC50 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC50 27.9 mM at pH 8.0, where the addition of 0.5 M NaCl altered the inhibition to the level observed at pH 9.8. APs from E. coli and calf intestines were less sensitive to cyclohexylamine, whereas an Antarctic bacterial AP was similar to VAP in this respect. X-ray crystallography at 2.3 Å showed two binding sites, one in the active site channel and another at the surface close to dimer interface. Antarctic bacterial AP and VAP have Trp274 in common in their active-sites, that takes part in binding cyclohexylamine. VAP variants W274A, W274K, and W274H gave IC50 values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8. Conclusions - The binding of cyclohexylamine in locations at the dimeric interface and/or in the active site of APs may delay product release or reduce the rate of catalytic step(s) involving conformational changes and intersubunit communications. General significance - Cyclohexylamine is a common chemical in industries and used as a counterion in substrates for alkaline phosphatase, a clinically important and common enzyme in the biosphere
Crystal and solution structures reveal oligomerization of individual capsid homology domains of Drosophila Arc
Abstract
Synaptic plasticity is vital for brain function and memory formation. One of the key proteins in long-term synaptic plasticity and memory is the activity-regulated cytoskeleton-associated protein (Arc). Mammalian Arc forms virus-like capsid structures in a process requiring the N-terminal domain and contains two C-terminal lobes that are structural homologues to retroviral capsids. Drosophila has two isoforms of Arc, dArc1 and dArc2, with low sequence similarity to mammalian Arc, but lacking a large N-terminal domain. Both dArc isoforms are related to the Ty3/gypsy retrotransposon capsid, consisting of N- and C-terminal lobes. Structures of dArc1, as well as capsids formed by both dArc isoforms, have been recently determined. We carried out structural characterization of the four individual dArc lobe domains. As opposed to the corresponding mammalian Arc lobe domains, which are monomeric, the dArc lobes were all oligomeric in solution, indicating a strong propensity for homophilic interactions. A truncated N-lobe from dArc2 formed a domain-swapped dimer in the crystal structure, resulting in a novel dimer interaction that could be relevant for capsid assembly or other dArc functions. This domain-swapped structure resembles the dimeric protein C of flavivirus capsids, as well as the structure of histones dimers, domain-swapped transcription factors, and membrane-interacting BAK domains. The strong oligomerization properties of the isolated dArc lobe domains explain the ability of dArc to form capsids in the absence of any large N-terminal domain, in contrast to the mammalian protein
Development and validation of Arc nanobodies:new tools for probing Arc dynamics and function
Abstract
Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions
Development and validation of Arc nanobodies:new tools for probing Arc dynamics and function
Abstract
Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions
Top hits from the nanobody CDR3 BLAST search.
The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. Both anti-Arc nanobodies bind specifically to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and allow to develop diagnostic and treatment tools.</div
Identification of a putative Arc-NL binding site on PICK1.
(A) AlphaFold2 model of the PICK1 BAR domain shows that the distal loop carries a PxY motif (pink). The side chains of the Pro and Tyr residues in the motif are shown. (B) Sequence alignment between the H11 CDR3 loop and the PxY motif of PICK1. Bold residues are identical, and the PxY motif is highlighted in red.</p