Caenorhabditis elegansCPB-1 (cytoplasmic polyadenylation element binding protein) and FBF (fem-3 mRNA binding factor) are evolutionary conserved regulators of mRNA translation that belong to the CPEB (cytoplasmic polyadenylation element binding) and PUF (Pumilio and FBF) protein families, respectively. In hermaphrodite worms CPB-1 and FBF control key steps during germline development, including stem cell maintenance and sex determination. While CPB-1 and FBF are known to interact, the molecular basis and function of the CPB-1•FBF complex are not known. The surface of CPB-1 that interacts with FBF was localized using in vivo and in vitro methods to a ten-residue region at the N-terminus of the protein and these residues are present in the FBF-binding protein GLD-3 (germline development defective). PUF proteins are characterized by the presence of eight α-helical repeats (PUF repeats) arranged side by side in an elongated structure. Critical residues for CPB-1 binding are found in the extended loop that connects PUF repeats seven and eight. The same FBF residues also mediate binding to GLD-3, indicating a conserved binding mode between different protein partners. CPB-1 binding was competitive with GLD-3, suggestive of mutual exclusivity in vivo. RNA binding measurements demonstrated that CPB-1 alters the affinity of FBF for specific RNA sequences, implying a functional model where the coregulatory protein CPB-1 modulates FBF target selection.
Pumilio; PUF protein; FBF; protein-protein interactions; germline development
The ribosome is an essential and highly complex biological system in all living cells. A large body of literature is available on the assembly of the ribosome in vitro, but a clear picture of this process inside the cell has yet to emerge. Here, we directly characterized in vivo ribosome assembly intermediates and associated assembly factors from wild-type E. coli cells using a general quantitative mass spectrometry (qMS) approach. The presence of distinct populations of ribosome assembly intermediates was verified using an in vivo stable isotope pulse-labeling approach, and their exact ribosomal protein (r-protein) contents were characterized against an isotopically labeled standard. The model-free clustering analysis of the resultant protein levels for the different ribosomal particles produced four 30S assembly groups that correlate very well with previous in vitro assembly studies of the small ribosomal subunit, and six 50S assembly groups that clearly define an in vivo assembly landscape for the larger ribosomal subunit. In addition, de novo proteomics identified a total of 21 known and potentially new ribosome assembly factors co-localized with various ribosomal particles. These results represent new in vivo assembly maps of the E. coli 30S and 50S subunits, and the general qMS approach should be a solid platform for future studies of ribosome biogenesis across a host of model organisms.
ribosome assembly; ribosome biogenesis; quantitative mass spectrometry; stable isotope pulse-labeling; ribosome assembly factors
The Pumilio and FBF (PUF) family of RNA-binding proteins interacts with protein partners to post-transcriptionally regulate mRNAs in eukaryotes. The interaction between PUF family member fem-3binding factor (FBF) and germline development defective-3 protein (GLD-3) promotes spermatogenesis in Caenorhabditis elegans by increasing expression of the fem-3 mRNA. Defined here in these studies is the molecular basis for this critical interaction. A 10 amino acid region within GLD-3 is required for FBF binding, while a 7 amino acid loop in FBF between PUF repeats 7 and 8 is necessary for GLD-3 binding. These short sequences are conserved, as other FBF-bindingproteins bear sequences similar to those in GLD-3 and other C. elegans PUF proteins contain sequences similar to those in FBF. The FBF-binding region of GLD-3 forms a ternary complex with FBF on the point mutation element (PME) in the fem-3 3’ untranslated region (UTR) and formation of this GLD-3•FBF complex does not impact the RNA-binding activity of FBF. These data raise the possibility of alternative models involving the formation of a GLD-3•FBF•RNA complex in the regulation of germline mRNAs.
RNA-binding proteins; PUF protein; fem-3; RNA•protein interactions; germline development
Coilin is widely known as the protein marker of the Cajal body, a subnuclear domain important to the biogenesis of small nuclear ribonucleoproteins and telomerase, complexes that are crucial to pre-messenger RNA splicing and telomere maintenance, respectively. Extensive studies have characterized the interaction between coilin and the various other protein components of CBs and related subnuclear domains, however only a few have examined interactions between coilin and nucleic acid. We have recently published that coilin is tightly associated with nucleic acid, displays RNase activity in vitro, and isre-distributed to the rRNA-rich nucleoli in cells treated with the DNA damaging agentscisplatin or etoposide. Here, we report a specific in vivo association between coilin and ribosomal RNA (rRNA), U small nuclear RNA (snRNA) and human telomerase RNA (hTR), which is altered upon treatment with DNA damaging agents. Using chromatin IP (ChIP), we provide evidence of coilin interaction with specific regions of U snRNA gene loci. We have also utilized bacterially expressed coilin fragments in order to map the region(s) important for RNA binding and RNase activity in vitro. Additionally, we provide evidence of coilin involvement in the processing of hTR both in vitro and in vivo.
Cajal body; SMN; telomere; splicing; nucleolus
Our laboratory has reported earlier that in leukocytes, phospholipase D2 (PLD2) is under control of Janus Kinase-3 (JAK3), which mediates chemotaxis. Investigating JAK3 in cancer cells led to an important discovery as exponentially growing MDA-MB-231 human breast cancer cells, which are highly proliferative and metastatic, did not substantially use JAK3 to activate PLD2. However, in 2-h or 16-h starved cell cultures, JAK3 switches to a PLD2-enhancing role, consistent with the needs of those cells to enter a “survival state” that relies on an increase in PLD2 activity to withstand serum deprivation. Using a small-molecule tyrosine kinase inhibitor, the flavonoid apigenin (4’,5,7-trihydroxyflavone), as well as RNA silencing, we found that the invasive phenotype of MDA-MB-231 cells is mediated by PLD2 under direct regulation of both JAK3 and the tyrosine kinase, Epidermal Growth Factor Receptor (EGFR). Further, serum-deprived cells in culture show an upregulated EGFR/JAK3/PLD2-PA system and are especially sensitive to a combination of JAK3 and PLD2 enzymatic activity inhibitors (30 nM apigenin and 300 nM 5-Fluoro-2-Indolyl des-Chlorohalopemide [FIPI], respectively). Thus, a multi-layered activation of cell invasion by two kinases (EGFR and JAK3) and a pholspholipase (PLD2) provides regulatory flexibility and maximizes the aggressively invasive power of MDA-MB-231 breast cancer cells. This is especially important in the absence of growth factors in serum, coincidental with migration of these cells to new locations.
Potent antiretroviral therapy (ART) has transformed HIV-1 infection into a chronic manageable disease; however drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study we utilized a combination of structure based analysis of Cyclin/CDK complexes with our previously published Tat peptide derivatives. We modeled the Tat peptide inhibitors with CDKs and found a particular pocket which showed the most stable binding site (Cavity 1) using in silico analysis. Furthermore, we were able to find peptide mimetics that bound to similar regions using in silico searches of a chemical library, followed by cell based biological assays. Using these methods we obtained the first generation mimetic drugs and tested these compounds on HIV-1 LTR activated transcription. Using biological assays followed by similar in silico analysis to find a 2nd generation drugs resembling the original mimetic, we found the new targets of Cavity 1 and Cavity 2 regions on CDK9. We examined the 2nd generation mimetic against various viral isolates, and observed a generalized suppression of most HIV-1 isolates. Finally, the drug inhibited viral replication in humanized mouse models of Rag2-/-γc-/- with no toxicity to the animals at tested concentrations. Our results suggest that it may be possible to model peptide inhibitors into available crystal structures and further find drug mimetics using in silico analysis.
ATP analog; CDK9; Cyclin T1; viral transactivator; inhibitors
A synthetic phage-displayed antibody repertoire was constructed with
equivalent chemical diversity in the third complementarity-determining regions
of the heavy (CDR-H3) and light chains (CDR-L3), which contrasts with natural
antibodies in which CDR-H3 is much more diverse than CDR-L3 due to the genetic
mechanisms that generate antibody encoding genes. Surprisingly, the synthetic
repertoire yielded numerous functional antibodies that contained mutated CDR-L3
sequences but a fixed CDR-H3 sequence. Alanine-scanning analysis of antibodies
that recognized ten different antigens but contained a common CDR-H3 loop showed
that, in most cases, the fixed CDR-H3 sequence was able to contribute favorably
to antigen recognition, but in some cases, the loop was functionally inert.
Structural analysis of one such antibody in complex with antigen showed that the
inert CDR-H3 loop was nonetheless highly buried at the antibody-antigen
interface. Taken together, these results show that CDR-H3 diversity is not
necessarily required for the generation of antibodies that recognize diverse
protein antigens with high affinity and specificity, and if given the chance,
CDR-L3 readily assumes the dominant role for antigen recognition. These results
contrast with the commonly accepted view of antigen recognition derived from the
analysis of natural antibodies, in which CDR-H3 is presumed to be dominant and
CDR-L3 is presumed to play an auxiliary role. Furthermore, the results show that
natural antibody function is genetically constrained, and it should be possible
to develop more functional synthetic antibody libraries by expanding the
diversity of CDR-L3 beyond what is observed in nature.
antibody library; phage display; protein engineering; affinity; specificity
We show that negative-stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one light chain or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and the essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~ 23 pN·nm/rad2 is estimated on average for all S1 types, similar to myosin-5. This translates to apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor–lever junction.
•Elasticity of muscle crossbridges is important, but its structural basis is obscure.•Muscle myosin heads from rabbit, scallop and chicken share a common structure.•The lever domain hinges about its connection with the motor domain.•The stiffness of the motor–lever hinge is lower than estimates for crossbridges.•Flexibility within the myosin head can be the basis of crossbridge stiffness.
S1, myosin subfragment-1; CkS1, chicken skeletal muscle myosin subfragment-1; mCkS1, methylated chicken skeletal muscle myosin subfragment-1; RbS1, rabbit skeletal muscle myosin subfragment-1; ScS1, scallop cross-striated adductor muscle myosin subfragment-1; MD, motor domain; ELC, essential light chain; RLC, regulatory light chain; EM, electron microscopy; 3D, three-dimensional; 2D, two-dimensional; electron microscopy; image processing; stiffness; pliant region; crossbridge
Activation of platelets by the serine protease thrombin is a critical event in haemostasis. This process involves the binding of thrombin to glycoprotein Ibα (GpIbα) and cleavage of protease-activated receptors (PARs). The N-terminal extracellular domain of GpIbα contains an acidic peptide stretch that has been identified as the main thrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIbα binding, but it remains unclear how they are involved. This issue is of critical importance for the mechanism of platelet activation by thrombin. If both exosites bind to GpIbα, thrombin could potentially act as a platelet adhesion molecule or receptor dimerisation trigger. Alternatively, if only a single site is involved, GpIbα may serve as a cofactor for PAR-1 activation by thrombin. To determine the involvement of thrombin's two exosites in GpIbα binding, we employed the complementary methods of mutational analysis, binding studies, X-ray crystallography and NMR spectroscopy. Our results indicate that the peptide corresponding to the C-terminal portion of GpIbα and the entire extracellular domain bind exclusively to thrombin's exosite II. The interaction of thrombin with GpIbα thus serves to recruit thrombin activity to the platelet surface while leaving exosite I free for PAR-1 recognition.
•We analysed interactions of the platelet receptor GpIbα with thrombin using three complementary methods.•GpIbα exclusively binds to exosite II of thrombin.•Exosite I remains available for binding to other ligands.•GpIbα recruits thrombin to the platelet membrane as a cofactor for PAR-1 cleavage.
GpIbα, glycoprotein Ibα; PAR, protease-activated receptor; TM, thrombomodulin; LRR, leucine-rich repeat; PPACK, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone; SPR, surface plasmon resonance; TROSY, transverse relaxation optimised spectroscopy; PEG, polyethylene glycol; crystallography; exosite; haemostasis; NMR; platelet
Repeat proteins composed of tandem arrays of a short structural motif often mediate protein-protein interactions. Past efforts to design repeat protein-based molecular recognition tools have focused on the creation of templates from the consensus of individual repeats, regardless of their natural context. Such an approach assumes that all repeats are essentially equivalent. In this study we present the results of a ‘module-based’ approach, in which modules composed of tandem repeats are aligned to identify repeat-specific features. Using this approach to analyze tetratricopeptide repeat modules that contain 3 tandem repeats (3TPRs), we identify two classes of 3TPR modules with distinct structural signatures that are correlated with different sets of functional residues. Our analyses also reveal a high degree of correlation between positions across the entire ligand-binding surface, indicative of a coordinated, coevolving binding surface. Extension of our analyses to different repeat protein modules reveals more examples of repeat-specific features, especially in armadillio repeat (ARM) modules. In summary, the module-based analyses that we present effectively capture key repeat-specific features that will be important to include in future repeat protein design templates.
The rugged protein sequence–function landscape complicates efforts, both in nature and in the laboratory, to evolve protein function. Protein library diversification must strike a balance between sufficient variegation to thoroughly sample alternative functionality versus the probability of mutant destabilization below an expressible threshold. In this work, we explore the sequence–function landscape in the context of screening for molecular recognition from an Ig scaffold library. The fibronectin type III domain is used to explore the impact of two sequence diversification strategies: (a) partial wild-type conservation at structurally important positions within the paratope region and (b) tailored amino acid composition mimicking antibody binding-site composition at putative paratope positions. Structurally important positions within the paratope region were identified through stability, structural, and phylogenetic analyses and partially or fully conserved in sequence. To achieve tailored antibody-like diversity, we designed a set of skewed nucleotide mixtures yielding codons approximately matching the distribution observed in antibody complementarity-determining regions without incurring the expense of triphosphoramidite-based construction. These design elements were explored via comparison of three library designs: a random library, a library with wild-type bias in the DE loop only and tyrosine–serine diversity elsewhere, and a library with wild-type bias at 11 positions and the antibody-inspired amino acid distribution. Using pooled libraries for direct competition in a single tube, selection and maturation of binders to seven targets yielded 19 of 21 clones that originated from the structurally biased, tailored-diversity library design. Sequence analysis of the selected clones supports the importance of both tailored compositional diversity and structural bias. In addition, selection of both well and poorly expressed clones from two libraries further elucidated the impact of structural bias.
fibronectin type III domain (Fn3); protein engineering; synthetic library; molecular recognition
The three-dimensional structures of the extended T4 phage tail and polysheath, an aberrant form of the contracted tail sheath, have both been reconstructed from electron micrographs. The reconstructed map of the extended structure is at a somewhat higher resolution (~20 Å) than an earlier reconstruction, allowing tentative boundaries to be drawn between the individual protein subunits in the tail sheath. This improvement has been achieved by testing the degree of correlation between data from different images as part of the selection procedure and averaging the most highly correlated sets of data. Details of the correlation are given in the Appendix. The resolution of the map of the contracted structure is lower than that of the extended tail, in spite of a similar averaging of several images, because of the higher degree of distortion in this structure. Nevertheless, it has been possible, to some extent, to trace the changes in conformation of the subunit during contraction.
Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.
Toll-like receptor 3; TLR3; innate immunity; lateral TLR3 clustering; quaternary complex
In eukaryotes, a variant of conventional histone H3 termed CenH3 epigenetically marks the centromere. The conserved CenH3 chaperone specifically recognizes CenH3 and is required for CenH3 deposition at the centromere. Recently, the structures of the chaperone/CenH3/H4 complexes have been determined for H. sapiens (Hs) and the budding yeasts S. cerevisiae (Sc) and K. lactis (Kl). Surprisingly, the three structures are very different, leading to different proposed structural bases for chaperone function. The question of which structural region of CenH3 provides the specificity determinant for the chaperone recognition is not fully answered. Here, we investigated these issues using solution NMR and site-directed mutagenesis. We discovered that, in contrast to previous findings, the structures of the Kl and Sc chaperone/CenH3/H4 complexes are actually very similar. This new finding reveals that both budding yeast and human chaperones use a similar structural region to block DNA from binding to the histones. Our mutational analyses further indicate that the N-terminal region of the CenH3 α2 helix is sufficient for specific recognition by the chaperone for both budding yeast and human. Thus, our studies have identified conserved structural bases of how the chaperones recognize CenH3 and perform the chaperone function.
The S. cerevisiae RNase III enzyme Rnt1p preferentially binds to dsRNA hairpin substrates with a conserved (A/u)GNN tetraloop fold, via shape-specific interactions by its dsRBD helix α1 to the tetraloop minor groove. To investigate whether conformational flexibility in the dsRBD regulates the binding specificity, we determined the backbone dynamics of the Rnt1p dsRBD in the free and AGAA hairpin-bound states using NMR spin relaxation experiments. The intrinsic μs-ms timescale dynamics of the dsRBD suggests that helix α1 undergoes conformational sampling in the free state, with large dynamics at some residues in the α1-β1 loop (α1-β1 hinge). To correlate free dsRBD dynamics with structural changes upon binding, we determined the solution structure of the free dsRBD used in the previously determined RNA-bound structures. The Rnt1p dsRBD has an extended hydrophobic core comprising helix α1, the α1-β1 loop, and helix α3. Analysis of the backbone dynamics and structures of the free and bound dsRBD reveals that slow-timescale dynamics in the α1-β1 hinge are associated with concerted structural changes in the extended hydrophobic core that govern binding of helix α1 to AGAA tetraloops. The dynamic behavior of the dsRBD bound to a longer AGAA hairpin reveals that dynamics within the hydrophobic core differentiate between specific and non-specific sites. Mutations of residues in the α1-β1 hinge result in changes to the dsRBD stability and RNA-binding affinity, and cause defects in snoRNA processing in vivo. These results reveal that dynamics in the extended hydrophobic core are important for binding site selection by the Rnt1p dsRBD.
NMR; Rnt1; RNA binding domain; protein-RNA; spin relaxation
Allostery in a protein involves effector binding at an allosteric site that changes the structure and/or dynamics at a distant, functional site. In addition to the chemical equilibrium of ligand binding, allostery involves a conformational equilibrium between one protein substate that binds the effector and a second substate that less strongly binds the effector. We run molecular dynamics simulations using simple, smooth energy landscapes to sample specific ligand-induced conformational transitions, as defined by the effector-bound and unbound protein structures. These simulations can be performed using our web server: http://salilab.org/allosmod/. We then develop a set of features to analyze the simulations and capture the relevant thermodynamic properties of the allosteric conformational equilibrium. These features are based on molecular mechanics energy functions, stereochemical effects, and structural/dynamic coupling between sites. Using a machine-learning algorithm on a dataset of 10 proteins and 179 mutations, we predict both the magnitude and sign of the allosteric conformational equilibrium shift by the mutation; the impact of a large identifiable fraction of the mutations can be predicted with an average unsigned error of 1 kBT. With similar accuracy, we predict the mutation effects for an 11th protein that was omitted from the initial training and testing of the machine-learning algorithm. We also assess which calculated thermodynamic properties contribute most to the accuracy of the prediction.
energy landscape; protein dynamics; machine learning; allostery
Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. H. pylori secretes a pore-forming exotoxin known as vacuolating toxin (VacA). VacA contains two distinct domains, designated p33 and p55, and assembles into large “snowflake”-shaped oligomers. Thus far, no structural data are available for the p33 domain, which is essential for membrane channel formation. Using single-particle electron microscopy and the random conical tilt approach, we have determined the three-dimensional structures of six VacA oligomeric conformations at ~15-Å resolution. The p55 domain, composed primarily of β-helical structures, localizes to the peripheral arms, while the p33 domain consists of two globular densities that localize within the center of the complexes. By fitting the VacA p55 crystal structure into the electron microscopy densities, we have mapped inter-VacA interactions that support oligomerization. In addition, we have examined VacA variants/mutants that differ from wild-type (WT) VacA in toxin activity and/or oligomeric structural features. Oligomers formed by VacAΔ6–27, a mutant that fails to form membrane channels, lack an organized p33 central core. Mixed oligomers containing both WT and VacAΔ6–27 subunits also lack an organized core. Oligomers formed by a VacA s2m1 chimera (which lacks cell-vacuolating activity) and VacAΔ301–328 (which retains vacuolating activity) each contain p33 central cores similar to those of WT oligomers. By providing the most detailed view of the VacA structure to date, these data offer new insights into the toxin's channel-forming component and the intermolecular interactions that underlie oligomeric assembly.
pore-forming toxins; single-particle electron microscopy; oligomerization; pathogenesis
Amyloid formation plays an important role in a broad range of diseases and the search for amyloid inhibitors is an active area of research. Amyloid formation takes places in a heterogeneous environment in vivo with the potential for interactions with membranes and with components of the extracellular matrix. Naturally occurring amyloid deposits are associated with sulfated proteoglycans and other factors. However, the vast majority of in vitro assays of amyloid formation and amyloid inhibition are conducted in homogeneous solution where the potential for interactions with membranes or sulfated proteoglycans is lacking and it is possible that different results may be obtained in heterogeneous environments. We show that variants of islet amyloid polypeptide, which are non-amyloidgenic in homogeneous solution, can be readily induced to form amyloid in the presence of glycosaminoglycans. Glycosaminoglycans are found to be more effective than anionic lipid vesicles at inducing amyloid formation on a per charge basis. Several known inhibitors of IAPP amyloid formation are shown to be less effective in the presence of glycosaminoglycans.
IAPP; amylin; glycosaminoglycan; extracellular matrix; amyloid; inhibitor
The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) translocates DNA and protein substrates across the bacterial cell envelope. Six presumptive channel subunits of this T4SS (VirD4, VirB11, VirB6, VirB8, VirB2, and VirB9) form close contacts with the VirD2-T-strand transfer intermediate during export, as shown recently by a novel transfer DNA immunoprecipitation (TrIP) assay. Here, we characterize the contribution of the hydrophobic channel component VirB6 to substrate translocation. Results of reporter protein fusion and cysteine accessibility studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, five transmembrane segments, and a cytoplasmic C terminus. TrIP studies aimed at characterizing the effects of VirB6 insertion and deletion mutations on substrate translocation identified several VirB6 functional domains: (i) a central region composed of a large periplasmic loop (P2) (residues 84 to 165) mediates the interaction of VirB6 with the exiting T-strand; (ii) a multi-membrane-spanning region carboxyl-terminal to loop P2 (residues 165 to 245) is required for substrate transfer from VirB6 to the bitopic membrane subunit VirB8; and (iii) the two terminal regions (residues 1 to 64 and 245 to 290) are required for substrate transfer to the periplasmic and outer membrane-associated VirB2 and VirB9 subunits. Our findings support a model whereby the periplasmic loop P2 comprises a portion of the secretion channel and distinct domains of VirB6 participate in channel subunit interactions required for substrate passage to the cell exterior.
conjugation; pathogenesis; secretion; DNA translocation; membrane protein topology
The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded transition intermediate of LacY, which would prevent leaking of the proton gradient. As no experimental structure is known, the conformational transition is not fully understood in atomic detail. We simulated transition events from a cytoplasmic open conformation to a periplasmic open conformation with the dynamic importance sampling molecular dynamics method and observed occluded intermediates. Analysis of water permeation pathways and the electrostatic free-energy landscape of a solvated proton indicated that the occluded state contains a solvated central cavity inaccessible from either side of the membrane. We propose a pair of geometric order parameters that capture the state of the pathway through the MFS transporters as shown by a survey of available crystal structures and models. We present a model for the occluded state of apo-LacY, which is similar to the occluded crystal structures of the MFS transporters EmrD, PepTSo, NarU, PiPT and XylE. Our simulations are consistent with experimental double electron spin–spin distance measurements that have been interpreted to show occluded conformations. During the simulations, a salt bridge that has been postulated to be involved in driving the conformational transition formed. Our results argue against a simple rigid-body domain motion as implied by a strict “rocker-switch mechanism” and instead hint at an intricate coupling between two flexible gates.
•The transport mechanism of LacY is hypothesized to involve an intermediate “occluded” state.•Such a state is observed in computer simulations of the conformational transitions.•Simulation data are validated with experimental double electron–electron spin resonance measurements.•The structural gating elements of LacY are identified.•Occluded LacY is similar to known occluded structures of homologous proteins.
DEER, double electron–electron spin resonance; DIMS, dynamic importance sampling; MD, molecular dynamics; MFS, major facilitator superfamily; MFS, major facilitator superfamily; POPC, 1-palmitoyl,2-oleoyl-sn-glycero-3-phosphocholine; SGLD, self-guided Langevin dynamics; 3D, three-dimensional; major facilitator superfamily transporters; molecular dynamics simulations; protein conformational change; membrane permeation; protons
Human IgG4, normally the least abundant of the four subclasses of IgG in serum, displays a number of unique biological properties. It can undergo heavy-chain exchange, also known as Fab-arm exchange, leading to the formation of monovalent but bispecific antibodies, and it interacts poorly with FcγRII and FcγRIII, and complement. These properties render IgG4 relatively “non-inflammatory” and have made it a suitable format for therapeutic monoclonal antibody production. However, IgG4 is also known to undergo Fc-mediated aggregation and has been implicated in auto-immune disease pathology. We report here the high-resolution crystal structures, at 1.9 and 2.35 Å, respectively, of human recombinant and serum-derived IgG4-Fc. These structures reveal conformational variability at the CH3–CH3 interface that may promote Fab-arm exchange, and a unique conformation for the FG loop in the CH2 domain that would explain the poor FcγRII, FcγRIII and C1q binding properties of IgG4 compared with IgG1 and -3. In contrast to other IgG subclasses, this unique conformation folds the FG loop away from the CH2 domain, precluding any interaction with the lower hinge region, which may further facilitate Fab-arm exchange by destabilisation of the hinge. The crystals of IgG4-Fc also display Fc–Fc packing contacts with very extensive interaction surfaces, involving both a consensus binding site in IgG-Fc at the CH2–CH3 interface and known hydrophobic aggregation motifs. These Fc–Fc interactions are compatible with intact IgG4 molecules and may provide a model for the formation of aggregates of IgG4 that can cause disease pathology in the absence of antigen.
•The first high-resolution crystal structures of IgG4-Fc have been solved.•Arg409 adopts two conformations, each with a different effect on the CH3–CH3 interface.•Crystal packing analysis reveals a novel Fc–Fc interface.•The CH2 domain FG loop adopts a unique conformation, affecting FcγR and C1q binding.•The IgG4-Fc crystal structures explain unique biological properties of IgG4.
FAE, Fab-arm exchange; IgG4-RD, IgG4-related disease; rFc, recombinant IgG4-Fc; sdFc, serum-derived IgG4-Fc; MES, 4-morpholineethanesulfonic acid; antibody; immunoglobulin; Fab-arm exchange; Fc receptor; C1q
Chromatin “remodeling” is widely accepted as the mechanism that permits access to DNA by the transcription machinery. To date, however, there has been no experimental measurement of the changes in the kinetics and thermodynamics of the DNA–histone octamer association that are required to remodel chromatin so that transcription may occur. Here, we present the results of optical tweezer measurements that compare the kinetic and thermodynamic properties of nucleosomes composed of unmodified histones with those of nucleosomes that contain a mutant histone H4 (H4-R45H), which has been shown to allow SWI/SNF remodeling factor-independent transcription from the yeast HO promoter in vivo. Our measurements, carried out in a force-clamp mode, determine the force-dependent unwinding and rewinding rates of the nucleosome inner turn. At each force studied, nucleosomes containing H4-R45H unwind more rapidly and rewind more slowly than nucleosomes containing unmodified H4, indicating that the latter are the more stable. Extrapolation to forces at which the winding and unwinding rates are equal determines the absolute free energy of the nucleosome inner turn to be −32kBT for nucleosomes containing unmodified H4 and −27kBT for nucleosomes containing H4-R45H. Thus, the “loosening” or “remodeling” caused by this point mutation, which is demonstrated to be sufficient to allow transcriptional machinery access to the HO promoter (in the absence of other remodeling factors), is 5kBT. The correlation between the free energy of the nucleosome inner turn and the sin (SWI/SNF-independent) transcription suggests that, beyond partial unwinding, complete histone unwinding may play a role in transcriptional activation.
chromatin; single molecule; optical tweezers; free energy; nucleosome
Papillomavirus (PV) E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here we show that recombinant E6 proteins from eight human and one bovine PV strains exist as oligomeric as well as multimeric species. These species were characterized using a variety of biochemical and biophysical techniques including analytical gel filtration, activity assays, SPR, EM and FTIR. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein (MBP), which slows down the formation of higher-order multimeric species. The proportion of each oligomeric form vary depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal HPV E6, retain binding to the ubiquitin ligase E6AP and the capacity to degrade the pro-apoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process which is accompanied by a significant increase in the β-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggest that self-association is a general property of E6 proteins which occurs both in vitro and in vivo and might therefore be functionally relevant.
HPV; E6; cervical cancer; self-association; oligomerization; ordered aggregation
Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer’s disease (AD). Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36-Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31–Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31–35 and residues 38–42. In contrast, Aβ(31–40) mainly existed as a statistical coil. To study the system experimentally, Aβ peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin were chemically synthesized. The triple substitution Gly33Val–Val36Pro–Gly38Val (“VPV”) facilitated Aβ42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild type Aβ42. When substituted into Aβ40, the VPV substitution caused the peptide to oligomerize similarly to Aβ42. The modified Aβ40 was significantly more toxic than Aβ40. The double substitution D-Pro36-L-Pro37 abolished hexamer and dodecamer formation by Aβ42 and produced an oligomer size distribution similar to that of Aβ40. Our data suggest that the Val36-Gly37 turn could be the sine qua non of Aβ42. If true, this structure would be an exceptionally important therapeutic target.
Amyloid β-protein; Alzheimer’s disease; β-hairpin