RÉSUMÉ
After more than two decades of research and development, adeno-associated virus (AAV) has become one of the dominant delivery vectors in gene therapy. Despite the focused research, the cell entry pathway for AAV is still not fully understood. Universal AAV receptor (AAVR) has been identified to be involved in cellular entry of different AAV serotypes. With the unveiling of the high-resolution AAV-AAVR complex structure by cryogenic electron microscopy, the atomic level interaction between AAV and AAVR has become the focus of study in recent years. However, the serotype dependence of this binding interaction and the effect of pH have not been studied. Here, orthogonal approaches including bio-layer interferometry (BLI), size-exclusion chromatography coupled to multi-angle laser scattering (SEC-MALS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) were utilized to study the interaction between selected AAV serotypes and AAVR under different pH conditions. A robust BLI method was developed and the equilibrium dissociation binding constants (KD) between different AAV serotypes (AAV1, AAV5 and AAV8) and AAVR was measured. The binding constants measured by BLI together with orthogonal methods (SEC-MALS and SV-AUC) all confirmed that AAV5 has the strongest binding affinity followed by AAV1 while AAV8 binds the weakest. It was also observed that lower pH promotes the binding between AAV and AAVR and neutral or slightly basic conditions lead to very weak binding. These data indicate that for certain serotypes, AAVR may play a prominent role in trafficking AAV to the Golgi rather than acting as a host cell receptor. Information obtained from these combinatorial biophysical methods can be used to engineer future generations of AAVs to have better transduction efficiency.
Sujet(s)
Dependovirus , Dependovirus/génétique , Dependovirus/composition chimique , Concentration en ions d'hydrogène , Liaison aux protéines , SérogroupeRÉSUMÉ
PURPOSE: Host cell proteins (HCPs) are impurities derived from expression systems during the manufacturing of biotherapeutics. Even trace amounts of certain HCPs can potentially compromise product safety and quality. Therefore, comprehensive analytical characterization is necessary. In particular, understanding how each HCP co-purifies with the biotherapeutics throughout the purification process would help guide process development to avoid further contamination. METHODS: We developed a new strategy based on size exclusion chromatography (SEC) fractionation followed by mass spectrometry (MS) analysis to study HCPs. RESULTS: Through an optimized experimental procedure, HCPs were effectively separated from monoclonal antibody (mAb) drug substances via SEC fractionation and sensitively detected with MS. Many HCPs were enriched in the high molecular weight fraction, thus indicating the formation of HCP-mAb complexes. SEC separation under mild denaturing conditions was demonstrated to disrupt weak interactions between certain HCPs and mAbs. The binding profiles of HCPs to mAbs were further characterized through comparison of the relative abundance of HCPs in each fraction under either native or mild denaturing SEC conditions. CONCLUSIONS: This new method not only achieves improved identification of HCPs in biotherapeutic drug substances but also offers an effective means to evaluate the binding properties between biotherapeutics and a wide range of HCPs.
Sujet(s)
Anticorps monoclonaux , Contamination de médicament , Animaux , Cricetinae , Anticorps monoclonaux/composition chimique , Chromatographie sur gel , Spectrométrie de masse/méthodes , Cricetulus , Cellules CHORÉSUMÉ
Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.
Sujet(s)
Régulation de l'expression des gènes , Peptide hydrolases/composition chimique , Tératogènes/composition chimique , Thalidomide/composition chimique , Facteurs de transcription/composition chimique , Protéines adaptatrices de la transduction du signal , Animaux , Protéines de liaison à l'ADN/composition chimique , Protéines de liaison à l'ADN/génétique , Homozygote , Humains , Immunohistochimie , Cellules souches pluripotentes induites , Ligands , Mâle , Souris , Souris transgéniques , Mutation , Protéines de tissu nerveux/composition chimique , Protéines de tissu nerveux/génétique , Peptide hydrolases/génétique , Protéolyse , Lapins , Testicule/métabolisme , Facteurs de transcription/génétique , Ubiquitin-protein ligases/métabolisme , Doigts de zincRÉSUMÉ
The first structural representative of the domain of unknown function DUF2006 family, also known as Pfam family PF09410, comprises a lipocalin-like fold with domain duplication. The finding of the calycin signature in the N-terminal domain, combined with remote sequence similarity to two other protein families (PF07143 and PF08622) implicated in isoprenoid metabolism and the oxidative stress response, support an involvement in lipid metabolism. Clusters of conserved residues that interact with ligand mimetics suggest that the binding and regulation sites map to the N-terminal domain and to the interdomain interface, respectively.
Sujet(s)
Protéines bactériennes/composition chimique , Bases de données génétiques , Métabolisme lipidique , Nitrosomonas europaea/composition chimique , Séquence d'acides aminés , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Nitrosomonas europaea/métabolisme , Stress oxydatif , Structure tertiaire des protéines , Alignement de séquences , Similitude de séquences d'acides aminésRÉSUMÉ
SSO2064 is the first structural representative of PF01796 (DUF35), a large prokaryotic family with a wide phylogenetic distribution. The structure reveals a novel two-domain architecture comprising an N-terminal, rubredoxin-like, zinc ribbon and a C-terminal, oligonucleotide/oligosaccharide-binding (OB) fold domain. Additional N-terminal helical segments may be involved in protein-protein interactions. Domain architectures, genomic context analysis and functional evidence from certain bacterial representatives of this family suggest that these proteins form a novel fatty-acid-binding component that is involved in the biosynthesis of lipids and polyketide antibiotics and that they possibly function as acyl-CoA-binding proteins. This structure has led to a re-evaluation of the DUF35 family, which has now been split into two entries in the latest Pfam release (v.24.0).
Sujet(s)
Acyl coenzyme A/composition chimique , Protéines d'archée/composition chimique , Pliage des protéines , Sulfolobus solfataricus/composition chimique , Zinc/composition chimique , Séquence d'acides aminés , Protéines d'archée/génétique , Protéines d'archée/métabolisme , Cristallographie aux rayons X , Génome d'archéobactérie , Modèles moléculaires , Données de séquences moléculaires , Liaison aux protéines , Structure tertiaire des protéines , Sulfolobus solfataricus/génétique , Sulfolobus solfataricus/métabolismeRÉSUMÉ
The crystal structure of Dhaf4260 from Desulfitobacterium hafniense DCB-2 was determined by single-wavelength anomalous diffraction (SAD) to a resolution of 2.01â Å using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). This protein structure is the first representative of the PF04016 (DUF364) Pfam family and reveals a novel combination of two well known domains (an enolase N-terminal-like fold followed by a Rossmann-like domain). Structural and bioinformatic analyses reveal partial similarities to Rossmann-like methyltransferases, with residues from the enolase-like fold combining to form a unique active site that is likely to be involved in the condensation or hydrolysis of molecules implicated in the synthesis of flavins, pterins or other siderophores. The genome context of Dhaf4260 and homologs additionally supports a role in heavy-metal chelation.
Sujet(s)
Protéines bactériennes/composition chimique , Desulfitobacterium/composition chimique , Métaux lourds/composition chimique , Enolase/composition chimique , Pliage des protéines , Séquence d'acides aminés , Protéines bactériennes/métabolisme , Domaine catalytique , Cristallographie aux rayons X , Desulfitobacterium/métabolisme , Métaux lourds/métabolisme , Modèles moléculaires , Données de séquences moléculaires , Liaison aux protéines , Structure tertiaire des protéinesRÉSUMÉ
Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (â¼40-99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.
Sujet(s)
Protéines bactériennes/composition chimique , Régulation de l'expression des gènes , Neisseria gonorrhoeae/composition chimique , Transcription génétique , Séquence d'acides aminés , Protéines bactériennes/génétique , Cristallographie aux rayons X , Génome bactérien , Modèles moléculaires , Données de séquences moléculaires , Neisseria gonorrhoeae/génétique , Structure quaternaire des protéines , Structure tertiaire des protéines , Similitude structurale de protéinesRÉSUMÉ
The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1â Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.
Sujet(s)
Acides aminés/métabolisme , Bordetella bronchiseptica/enzymologie , Chorismate mutase/composition chimique , Pliage des protéines , Rhodobacteraceae/enzymologie , Séquence d'acides aminés , Bacillus/enzymologie , Chorismate mutase/métabolisme , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Structure quaternaire des protéines , Structure tertiaire des protéines , Similitude structurale de protéinesRÉSUMÉ
The crystal structure of Jann_2411 from Jannaschia sp. strain CCS1, a member of the Pfam PF07336 family classified as a domain of unknown function (DUF1470), was solved to a resolution of 1.45â Å by multiple-wavelength anomalous dispersion (MAD). This protein is the first structural representative of the DUF1470 Pfam family. Structural analysis revealed a two-domain organization, with the N-terminal domain presenting a new fold called the ABATE domain that may bind an as yet unknown ligand. The C-terminal domain forms a treble-clef zinc finger that is likely to be involved in DNA binding. Analysis of the Jann_2411 protein and the broader ABATE-domain family suggests a role as stress-induced transcriptional regulators.
Sujet(s)
Protéines bactériennes/composition chimique , Rhodobacteraceae/composition chimique , Séquence d'acides aminés , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Structure quaternaire des protéines , Structure tertiaire des protéines , Alignement de séquences , Doigts de zincRÉSUMÉ
The structure of LP2179, a member of the PF08866 (DUF1831) family, suggests a novel α+ß fold comprising two ß-sheets packed against a single helix. A remote structural similarity to two other uncharacterized protein families specific to the Bacillus genus (PF08868 and PF08968), as well as to prokaryotic S-adenosylmethionine decarboxylases, is consistent with a role in amino-acid metabolism. Genomic neighborhood analysis of LP2179 supports this functional assignment, which might also then be extended to PF08868 and PF08968.
Sujet(s)
Acides aminés/métabolisme , Protéines bactériennes/composition chimique , Lactobacillus plantarum/composition chimique , Pliage des protéines , Séquence d'acides aminés , Protéines bactériennes/métabolisme , Cristallographie aux rayons X , Lactobacillus plantarum/métabolisme , Modèles moléculaires , Données de séquences moléculaires , Structure tertiaire des protéines , Alignement de séquences , Similitude structurale de protéinesRÉSUMÉ
The crystal structure of PA1994 from Pseudomonas aeruginosa, a member of the Pfam PF06475 family classified as a domain of unknown function (DUF1089), reveals a novel fold comprising a 15-stranded ß-sheet wrapped around a single α-helix that assembles into a tight dimeric arrangement. The remote structural similarity to lipoprotein localization factors, in addition to the presence of an acidic pocket that is conserved in DUF1089 homologs, phospholipid-binding and sugar-binding proteins, indicate a role for PA1994 and the DUF1089 family in glycolipid metabolism. Genome-context analysis lends further support to the involvement of this family of proteins in glycolipid metabolism and indicates possible activation of DUF1089 homologs under conditions of bacterial cell-wall stress or host-pathogen interactions.
Sujet(s)
Protéines bactériennes/composition chimique , Glycolipides/métabolisme , Pliage des protéines , Pseudomonas aeruginosa/composition chimique , Séquence d'acides aminés , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Cristallographie aux rayons X , Génome bactérien , Modèles moléculaires , Données de séquences moléculaires , Structure quaternaire des protéines , Structure tertiaire des protéines , Pseudomonas aeruginosa/génétique , Pseudomonas aeruginosa/métabolismeRÉSUMÉ
The crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.
Sujet(s)
Protéines bactériennes/composition chimique , Pliage des protéines , Rhodobacteraceae/composition chimique , Shewanella/composition chimique , Transduction du signal , Séquence d'acides aminés , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Cristallographie aux rayons X , Génome bactérien , Modèles moléculaires , Données de séquences moléculaires , Structure secondaire des protéines , Structure tertiaire des protéines , Rhodobacteraceae/génétique , Rhodobacteraceae/métabolisme , Shewanella/génétique , Shewanella/métabolisme , Similitude structurale de protéinesRÉSUMÉ
YeaZ is involved in a protein network that is essential for bacteria. The crystal structure of YeaZ from Thermotoga maritima was determined to 2.5â Å resolution. Although this protein belongs to a family of ancient actin-like ATPases, it appears that it has lost the ability to bind ATP since it lacks some key structural features that are important for interaction with ATP. A conserved surface was identified, supporting its role in the formation of protein complexes.
Sujet(s)
Protéines bactériennes/composition chimique , Thermotoga maritima/composition chimique , Séquence d'acides aminés , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Structure quaternaire des protéines , Structure tertiaire des protéines , Alignement de séquencesRÉSUMÉ
The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78â Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.
Sujet(s)
Bacillales/enzymologie , Pyrophosphatases/composition chimique , Séquence d'acides aminés , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Multimérisation de protéines , Structure quaternaire des protéines , Structure tertiaire des protéines , Similitude structurale de protéinesRÉSUMÉ
The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25â Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/ß SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the `open' conformation (YP_001095227.1), these helices are 15â Å apart, leading to the creation of a deep nonpolar cavity. In the `closed' structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their `open' monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.
Sujet(s)
Protéines bactériennes/composition chimique , Membrane cellulaire/composition chimique , Shewanella/composition chimique , Séquence d'acides aminés , Protéines bactériennes/métabolisme , Membrane cellulaire/métabolisme , Cristallographie aux rayons X , Ligands , Modèles moléculaires , Données de séquences moléculaires , Liaison aux protéines , Structure quaternaire des protéines , Structure tertiaire des protéines , Alignement de séquences , Similitude de séquences d'acides aminés , Shewanella/métabolisme , Similitude structurale de protéinesRÉSUMÉ
KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.
Sujet(s)
Protéines bactériennes/composition chimique , Klebsiella pneumoniae/composition chimique , Lipoprotéines/composition chimique , Séquence d'acides aminés , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Pliage des protéines , Structure tertiaire des protéinesRÉSUMÉ
Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a ß-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to ß-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins.
Sujet(s)
Bacteroides/composition chimique , Protéines périplasmiques/composition chimique , Séquence d'acides aminés , Bacteroides/métabolisme , Séquence conservée , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Protéines périplasmiques/métabolisme , Liaison aux protéines , Structure tertiaire des protéines , Alignement de séquences , Similitude structurale de protéinesRÉSUMÉ
The crystal structure of the Bacteroides thetaiotaomicron protein BT_3984 was determined to a resolution of 1.7â Å and was the first structure to be determined from the extensive SusD family of polysaccharide-binding proteins. SusD is an essential component of the sus operon that defines the paradigm for glycan utilization in dominant members of the human gut microbiota. Structural analysis of BT_3984 revealed an N-terminal region containing several tetratricopeptide repeats (TPRs), while the signature C-terminal region is less structured and contains extensive loop regions. Sequence and structure analysis of BT_3984 suggests the presence of binding interfaces for other proteins from the polysaccharide-utilization complex.
Sujet(s)
Protéines bactériennes/composition chimique , Bacteroides/composition chimique , Séquence d'acides aminés , Cristallographie aux rayons X , Modèles moléculaires , Données de séquences moléculaires , Structure tertiaire des protéines , Similitude structurale de protéinesRÉSUMÉ
BT1062 from Bacteroides thetaiotaomicron is a homolog of Mfa2 (PGN0288 or PG0179), which is a component of the minor fimbriae in Porphyromonas gingivalis. The crystal structure of BT1062 revealed a conserved fold that is widely adopted by fimbrial components.
Sujet(s)
Bacteroides/composition chimique , Protéines de fimbriae/composition chimique , Fimbriae bactériens/composition chimique , Pliage des protéines , Séquence d'acides aminés , Bacteroides/génétique , Cristallographie aux rayons X , Protéines de fimbriae/génétique , Modèles moléculaires , Données de séquences moléculaires , Structure tertiaire des protéines , Alignement de séquences , Similitude structurale de protéinesRÉSUMÉ
BT2081 from Bacteroides thetaiotaomicron (GenBank accession code NP_810994.1) is a member of a novel protein family consisting of over 160 members, most of which are found in the different classes of Bacteroidetes. Genome-context analysis lends support to the involvement of this family in carbohydrate metabolism, which plays a key role in B. thetaiotaomicron as a predominant bacterial symbiont in the human distal gut microbiome. The crystal structure of BT2081 at 2.05â Å resolution represents the first structure from this new protein family. BT2081 consists of an N-terminal domain, which adopts a ß-sandwich immunoglobulin-like fold, and a larger C-terminal domain with a ß-sandwich jelly-roll fold. Structural analyses reveal that both domains are similar to those found in various carbohydrate-active enzymes. The C-terminal ß-jelly-roll domain contains a potential carbohydrate-binding site that is highly conserved among BT2081 homologs and is situated in the same location as the carbohydrate-binding sites that are found in structurally similar glycoside hydrolases (GHs). However, in BT2081 this site is partially occluded by surrounding loops, which results in a deep solvent-accessible pocket rather than a shallower solvent-exposed cleft.