Characterization of size, anisotropy, and density heterogeneity of nanoparticles by sedimentation velocity.

A critical problem in materials science is the accurate characterization of the size dependent properties of colloidal inorganic nanocrystals. Due to the intrinsic polydispersity present during synthesis, dispersions of such materials exhibit simultaneous heterogeneity in density ρ, molar mass M, and particle diameter d. The density increments ∂ρ/∂d and ∂ρ/∂M of these nanoparticles, if known, can then provide important information about crystal growth and particle size distributions. For most classes of nanocrystals, a mixture of surfactants is added during synthesis to control their shape, size, and optical properties. However, it remains a challenge to accurately determine the amount of passivating ligand bound to the particle surface post synthesis. The presence of the ligand shell hampers an accurate determination of the nanocrystal diameter. Using CdSe and PbS semiconductor nanocrystals, and the ultrastable silver nanoparticle (M4Ag44(p-MBA)30), as model systems, we describe a Custom Grid method implemented in UltraScan-III for the characterization of nanoparticles and macromolecules using sedimentation velocity analytical ultracentrifugation. We show that multiple parametrizations are possible, and that the Custom Grid method can be generalized to provide high resolution composition information for mixtures of solutes that are heterogeneous in two out of three parameters. For such cases, our method can simultaneously resolve arbitrary two-dimensional distributions of hydrodynamic parameters when a third property can be held constant. For example, this method extracts partial specific volume and molar mass from sedimentation velocity data for cases where the anisotropy can be held constant, or provides anisotropy and partial specific volume if the molar mass is known.

Structure of the polycomb group protein PCGF1 in complex with BCOR reveals basis for binding selectivity of PCGF homologs.

Polycomb-group RING finger homologs (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6) are critical components in the assembly of distinct Polycomb repression complex 1 (PRC1)-related complexes. Here, we identify a protein interaction domain in BCL6 corepressor, BCOR, which binds the RING finger- and WD40-associated ubiquitin-like (RAWUL) domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals that (1) PUFD binds to the same surfaces as observed for a different Polycomb group RAWUL domain and (2) the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.

Human polyhomeotic homolog 3 (PHC3) sterile alpha motif (SAM) linker allows open-ended polymerization of PHC3 SAM.

Sterile alpha motifs (SAMs) are frequently found in eukaryotic genomes. An intriguing property of many SAMs is their ability to self-associate, forming an open-ended polymer structure whose formation has been shown to be essential for the function of the protein. What remains largely unresolved is how polymerization is controlled. Previously, we had determined that the stretch of unstructured residues N-terminal to the SAM of a Drosophila protein called polyhomeotic (Ph), a member of the polycomb group (PcG) of gene silencers, plays a key role in controlling Ph SAM polymerization. Ph SAM with its native linker created shorter polymers compared to Ph SAM attached to either a random linker or no linker. Here, we show that the SAM linker for the human Ph ortholog, polyhomeotic homolog 3 (PHC3), also controls PHC3 SAM polymerization but does so in the opposite fashion. PHC3 SAM with its native linker allows longer polymers to form compared to when attached to a random linker. Attaching the PHC3 SAM linker to Ph SAM also resulted in extending Ph SAM polymerization. Moreover, in the context of full-length Ph protein, replacing the SAM linker with PHC3 SAM linker, intended to create longer polymers, resulted in greater repressive ability for the chimera compared to wild-type Ph. These findings show that polymeric SAM linkers evolved to modulate a wide dynamic range of SAM polymerization abilities and suggest that rationally manipulating the function of SAM containing proteins through controlling their SAM polymerization may be possible.

The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain.

Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.

Construction and analyses of tetrameric forms of yeast NAD+-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. The crystal structure suggested that the interactions between tetramers in the octamer are restricted to defined regions in IDH1 subunits from each tetramer. Using truncation and mutagenesis, we constructed three tetrameric forms of IDH. Truncation of five residues from the amino terminus of IDH1 did not alter the octameric form of the enzyme, but this truncation with an IDH1 G15D or IDH1 D168K residue substitution produced tetrameric enzymes as assessed by sedimentation velocity ultracentrifugation. The IDH1 G15D substitution in the absence of any truncation of IDH1 was subsequently found to be sufficient for production of a tetrameric enzyme. The tetrameric forms of IDH exhibited ∼50% reductions in V(max) and in cooperativity with respect to isocitrate relative to those of the wild-type enzyme, but they retained the property of allosteric activation by AMP. The truncated (-5)IDH1/IDH2 and tetrameric enzymes were much more sensitive than the wild-type enzyme to inhibition by the oxidant diamide and concomitant formation of a disulfide bond between IDH2 Cys-150 residues. Binding of ligands reduced the sensitivity of the wild-type enzyme to diamide but had no effect on inhibition of the truncated or tetrameric enzymes. These results suggest that the octameric structure of IDH has in part evolved for regulation of disulfide bond formation and activity by ensuring the proximity of the amino terminus of an IDH1 subunit of one tetramer to the IDH2 Cys-150 residues in the other tetramer.

Dynamic interactions between clathrin and locally structured elements in a disordered protein mediate clathrin lattice assembly.

Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized ß-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.

Polycomb group targeting through different binding partners of RING1B C-terminal domain.

RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.

Disrupted zinc-binding sites in structures of pathogenic SOD1 variants D124V and H80R.

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we present structures of the pathogenic SOD1 variants D124V and H80R, both of which demonstrate compromised zinc-binding sites. The disruption of the zinc-binding sites in H80R SOD1 leads to conformational changes in loop elements, permitting non-native SOD1-SOD1 interactions that mediate the assembly of these proteins into higher-order filamentous arrays. Analytical ultracentrifugation sedimentation velocity experiments indicate that these SOD1 variants are more prone to monomerization than the wild-type enzyme. Although D124V and H80R SOD1 proteins appear to have fully functional copper-binding sites, inductively coupled plasma mass spectrometery (ICP-MS) and anomalous scattering X-ray diffraction analyses reveal that zinc (not copper) occupies the copper-binding sites in these variants. The absence of copper in these proteins, together with the results of covalent thiol modification experiments in yeast strains with and without the gene encoding the copper chaperone for SOD1 (CCS), suggests that CCS may not fully act on newly translated forms of these polypeptides. Overall, these findings lend support to the hypothesis that immature mutant SOD1 species contribute to toxicity in SOD1-linked ALS.

Characterization of reversible associations by sedimentation velocity with UltraScan.

We compare here the utility of sedimentation velocity (SV) to sedimentation equilibrium (SE) analysis for the characterization of reversible systems. Genetic algorithm optimization in UltraScan is used to optimize the model and to obtain solution properties of all components present in the system. We apply our method to synthetic and experimental data, and suggest limits for the accessible kinetic range. We conclude that equilibrium constants obtained from SV and SE analysis are equivalent, but that SV experiments provide better confidence for the K(d), can better account for the presence of contaminants and provide additional information including rate constants and shape parameters.

Design of thiolate rich metal binding sites within a peptidic framework.

A de novo protein design strategy provides a powerful tool to elucidate how heavy metals interact with proteins.Cysteine derivatives of the TRI peptide family (Ac-G(LKALEEK)4G-NH2) have been shown to bind heavy metals in an unusual trigonal geometry. Our present objective was to design binding sites in R-helical scaffolds that are able to form higher coordination number complexes with Cd(II) and Hg(II). Herein, we evaluate the binding of Cd(II) and Hg(II) to double cysteine substituted TRI peptides lacking intervening leucines between sulfurs in the heptads. We compare a -Cysd-X-X-X-Cysa- binding motif found in TRIL12CL16C to the more common -Cysa-X-X-Cysd- sequence of native proteins found in TRIL9CL12C. Compared to TRI, these substitutions destabilize the helical aggregates,leading to mixtures of two- and three-stranded bundles. The three-stranded coiled coils are stabilized by the addition of metals. TRIL9CL12C forms distorted tetrahedral complexes with both Cd(II) and Hg(II), as supported by UV-vis,CD, 113Cd NMR, 199Hg NMR and 111mCd PAC spectroscopy. Additionally, these signatures are very similar to those found for heavy metal substituted rubredoxin. These results suggest that in terms of Hg(II) binding, TRIL9CL12Ccan be considered as a good mimic of the metallochaperone HAH1, that has previously been shown to form protein dimers. TRIL12CL16C has limited ability to generate homoleptic tetrahedral complexes (Cd(SR)42-). These type of complexes were identified only for Hg(II). However, the spectroscopic signatures suggest a different geometry around the metal ion, demonstrating that effective metal sequestration into the hydrophobic interior of the bundle requires more than simply adding two sulfur residues in adjacent layers of the peptide core. Thus, proper design of metal binding sites must also consider the orientation of cysteine sidechains in a vs d positions of the heptads.

Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain.

Polycomb group (PcG) proteins are required for maintaining cell identity and stem cell self-renewal. RING1B and Polycomb (Pc) are two components of a multiprotein complex called polycomb repression complex 1 (PRC1) that is essential for establishing and maintaining long-term repressed gene states. Here we characterize the interaction between the C-terminal region of RING1B (C-RING1B) and the Pc cbox domain. The C-RING1B-cbox interaction displays a 1:1 stoichiometry with dissociation constants ranging from 9.2 to 180 nM for the different Pc orthologues. NMR analysis of C-RING1B alone reveals line broadening. However, when it is in complex with the cbox domain, there is a striking change to the NMR spectrum indicative of conformational tightening. This conformational change may arise from the organization of the C-RING1B subdomains. The C-terminal regions of all PcG RING1 proteins are composed of two stretches of conserved sequences separated by a variable linker sequence. While the entire C-RING1B region is required for cbox binding, the N- and C-terminal halves of C-RING1B can be separated and are able to interact, suggesting the presence of an intramolecular interaction within C-RING1B. The flexibility within the C-RING1B structure allowing transitions between the intramolecular bound and unbound states may cause the broadened peaks of the C-RING1B NMR spectrum. Binding the cbox domain stabilizes C-RING1B, whereby broadening is eliminated. The presence of flexible regions could allow C-RING1B to bind a variety of different factors, ultimately recruiting RING1B and its associated PcG proteins to different genomic loci.

The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site.

Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1(ts)) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 A resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 A resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 A resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase.

Reversible self-association of recombinant bovine factor B.

The recombinant bovine factor B, obtained by a newly developed bacterial expression system, was found to exhibit features characteristic of a reversible self-associating system. Using size-sieving chromatography, distribution of the factor B species ranged from a monomer to a trimer, but not oligomers of higher molecular weights. At high protein concentrations, factor B migrated as a single band in a native gel. Cross-linking with the amino-reactive cross-linking reagent bis (sulfosuccinimidyl) suberate (BS), at a low cross-linker to protein ratio yielded cross-linked products identified as factor B dimer and trimer. The cross-linking pattern was shown to be a function of the protein and cross-linker concentrations. The range of sedimentation coefficients in a sedimentation velocity experiment suggested that the largest particle present in the distribution was more than twice as large as the smallest. The data obtained under multiple conditions in the sedimentation equilibrium experiments are best fit to a model describing a reversible self-association of a monomer-trimer of factor B species, with a dissociation constant Kd(1,3)=2.48x10(-10) M(2).

Gene selection, alternative splicing, and post-translational processing regulate neuroligin selectivity for beta-neurexins.

Neuroligins 1-4 are postsynaptic transmembrane proteins capable of initiating presynaptic maturation via interactions with beta-neurexin. Both neuroligins and beta-neurexins have alternatively spliced inserts in their extracellular domains. Using analytical ultracentrifugation, we determined that the extracellular domains of the neuroligins sediment as dimers, whereas the extracellular domains of the beta-neurexins appear monomeric. Sedimentation velocity experiments of titrated stoichiometry ratios of beta-neurexin and neuroligin suggested a 2:2 complex formation. The recognition properties of individual neuroligins toward beta-neurexin-1 (NX1beta), along with the influence of their splice inserts, were explored by surface plasmon resonance and affinity chromatography. Different neuroligins display a range of NX1beta affinities spanning more than 2 orders of magnitude. Whereas splice insert 4 in beta-neurexin appears to act only as a modulator of the neuroligin/beta-neurexin association, splice insert B in neuroligin-1 (NL1) is the key element regulating the NL1/NX1beta binding. Our data indicate that gene selection, mRNA splicing, and post-translational modifications combine to give rise to a controlled neuroligin recognition code with a rank ordering of affinities for particular neurexins that is conserved for the neuroligins across mammalian species.

Solution formation of Holliday junctions in inverted-repeat DNA sequences.

The structure of Holliday junctions has now been well characterized at the atomic level through single-crystal X-ray diffraction in symmetric (inverted-repeat) DNA sequences. At issue, however, is whether the formation of these four-stranded complexes in solution is truly sequence dependent in the manner proposed or is an artifact of the crystallization process and, therefore, has no relevance to the behavior of this central intermediate in homologous recombination and recombination-dependent cellular processes. Here, we apply analytical ultracentrifugation to demonstrate that the sequence d(CCGGTACCGG), which crystallizes in the stacked-X form of the junction, assembles into four-stranded junctions in solution in a manner that is dependent on the DNA and cation concentrations, with an equilibrium established between the junction and duplex forms at 100-200 microM DNA duplex. In contrast, the sequence d(CCGCTAGCGG), which has been crystallized as B-DNA, is seen to adopt only the double-helical form at all DNA and salt concentrations that were tested. Thus, the ACC trinucleotide core is now shown to be important for the formation of Holliday junctions in both crystals and in solution and can be estimated to contribute approximately -4 kcal/mol to stabilizing this recombination intermediate in inverted-repeat sequences.

Structure and activation mechanism of the Drosophila initiator caspase Dronc.

Activation of an initiator caspase is essential to the execution of apoptosis. The molecular mechanisms by which initiator caspases are activated remain poorly understood. Here we demonstrate that the autocatalytic cleavage of Dronc, an important initiator caspase in Drosophila, results in a drastic enhancement of its catalytic activity in vitro. The autocleaved Dronc forms a homodimer, whereas the uncleaved Dronc zymogen exists exclusively as a monomer. Thus the autocatalytic cleavage in Dronc induces its stable dimerization, which presumably allows the two adjacent monomers to mutually stabilize their active sites, leading to activation. Crystal structure of a prodomain-deleted Dronc zymogen, determined at 2.5 A resolution, reveals an unproductive conformation at the active site, which is consistent with the observation that the zymogen remains catalytically inactive. This study revealed insights into mechanism of Dronc activation, and in conjunction with other observations, suggests diverse mechanisms for the activation of initiator caspases.

Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability.

The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.

T4 AsiA blocks DNA recognition by remodeling sigma70 region 4.

Bacteriophage T4 AsiA is a versatile transcription factor capable of inhibiting host gene expression as an 'anti-sigma' factor while simultaneously promoting gene-specific expression of T4 middle genes in conjunction with T4 MotA. To accomplish this task, AsiA engages conserved region 4 of Eschericia coli sigma70, blocking recognition of most host promoters by sequestering the DNA-binding surface at the AsiA/sigma70 interface. The three-dimensional structure of an AsiA/region 4 complex reveals that the C-terminal alpha helix of region 4 is unstructured, while four other helices adopt a completely different conformation relative to the canonical structure of unbound region 4. That AsiA induces, rather than merely stabilizes, this rearrangement can be realized by comparison to the homologous structures of region 4 solved in a variety of contexts, including the structure of Thermotoga maritima sigmaA region 4 described herein. AsiA simultaneously occupies the surface of region 4 that ordinarily contacts core RNA polymerase (RNAP), suggesting that an AsiA-bound sigma70 may also undergo conformational changes in the context of the RNAP holoenzyme.

Effect of AMP on mRNA binding by yeast NAD+-specific isocitrate dehydrogenase.

Yeast mitochondrial NAD+-specific isocitrate dehydrogenase (IDH) has previously been shown to bind specifically to 5'-untranslated regions of yeast mitochondrial mRNAs, and transcripts containing these regions have been found to allosterically inhibit activity of the enzyme. This inhibition is relieved by AMP, an allosteric activator of this regulatory enzyme of the tricarboxylic acid cycle. We further investigated these enzyme/ligand interactions to determine if binding of RNA and AMP by IDH is competitive or independent. Gel mobility shift experiments indicated no effect of AMP on formation of an IDH/RNA complex. Similarly, sedimentation velocity ultracentrifugation experiments used to analyze interactions in solution indicated that AMP alone had little effect on the formation or stability of an RNA/IDH complex. However, when these sedimentation experiments were conducted in the presence of isocitrate, which has been shown to be essential for binding of AMP by IDH, the proportion of RNA sedimenting in a complex with IDH was significantly reduced by AMP. These results suggest that AMP can affect the binding of RNA by IDH but that this effect is apparent only in the presence of substrate. They also suggest that the catalytic activity of IDH in vivo may be subject to complex allosteric control determined by relative mitochondrial concentrations of mRNA, isocitrate, and AMP. We also found evidence for binding of 5'-untranslated regions of mitochondrial mRNAs by yeast mitochondrial NADP+-specific isocitrate dehydrogenase (IDP1) but not by the corresponding cytosolic isozyme (IDP2). However, this appears to be a nonspecific interaction since no evidence was obtained for any effect on the catalytic activity of IDP1.