Visualizing Collagen Fibrils in the Cochlea's Tectorial and Basilar Membranes Using a Fluorescently Labeled Collagen-Binding Protein Fragment.

PURPOSE: A probe that binds to unfixed collagen fibrils was used to image the shapes and fibrous properties of the TM and BM. The probe (CNA35) is derived from the bacterial adhesion protein CNA. We present confocal images of hydrated gerbil TM, BM, and other cochlear structures stained with fluorescently labeled CNA35. A primary purpose of this article is to describe the use of the CNA35 collagen probe in the cochlea. METHODS: Recombinant poly-histidine-tagged CNA35 was expressed in Escherichia coli, purified by cobalt-affinity chromatography, fluorescence labeled, and further purified by gel filtration chromatography. Cochleae from freshly harvested gerbil bullae were irrigated with and then incubated in CNA35 for periods ranging from 2 h - overnight. The cochleae were fixed, decalcified, and dissected. Isolated cochlear turns were imaged by confocal microscopy. RESULTS: The CNA35 probe stained the BM and TM, and volumetric imaging revealed the shape of these structures and the collagen fibrils within them. The limbal zone of the TM stained intensely. In samples from the cochlear base, intense staining was detected on the side of the TM that faces hair cells. In the BM pectinate zone, staining was intense at the upper and lower boundaries. The BM arcuate zone was characterized by a prominent longitudinal collagenous structure. The spiral ligament, limbus and lamina stained for collagen, and within the spiral limbus the habenula perforata were outlined with intense staining. CONCLUSION: The CNA35 probe provides a unique and useful view of collagenous structures in the cochlea.

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments.

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu2+ from actin. Altogether, our data indicate that purified recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments.

Alterations to the broad-spectrum formin inhibitor SMIFH2 modulate potency but not specificity.

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.

Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4 + clusters under physiological conditions.

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn2+ and Cu+ binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn2+ binding was measured by chelation titrations of Zn7MT-3, while Cu+ binding was measured by Zn2+ displacement from Zn7MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant (K) and the change in enthalpy (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn2+ was also chelated from the individual α and ß domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn7MT-2 with Cu+ revealed that both MT isoforms have similar Cu+ affinities and binding thermodynamics, indicating that ΔH and ΔS are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu4 +-thiolate clusters when Cu+ displaces Zn2+ under physiological conditions. Comparison of the Zn2+ and Cu+ binding thermodynamics reveal that enthalpically-favoured Cu+, which forms Cu4 +-thiolate clusters, displaces the entropically-favoured Zn2+. These results provide a detailed thermodynamic analysis of d10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.

An alkane monooxygenase (AlkB) family in which all electron transfer partners are covalently bound to the oxygen-activating hydroxylase.

Alkane monooxygenase (AlkB) is a non-heme diiron enzyme that catalyzes the hydroxylation of alkanes. It is commonly found in alkanotrophic organisms that can live on alkanes as their sole source of carbon and energy. Activation of AlkB occurs via two-electron reduction of its diferric active site, which facilitates the binding, activation, and cleavage of molecular oxygen for insertion into an inert CH bond. Electrons are typically supplied by NADH via a rubredoxin reductase (AlkT) to a rubredoxin (AlkG) to AlkB, although alternative electron transfer partners have been observed. Here we report a family of AlkBs in which both electron transfer partners (a ferredoxin and a ferredoxin reductase) appear as an N-terminal gene fusion to the hydroxylase (ferr_ferrR_AlkB). This enzyme catalyzes the hydroxylation of medium chain alkanes (C6-C14), with a preference for C10-C12. It requires only NADH for activity. It is present in a number of bacteria that are known to be human pathogens. A survey of the genome neighborhoods in which is it found suggest it may be involved in alkane metabolism, perhaps facilitating growth of pathogens in non-host environments.

Variable Autoinhibition among Deafness-Associated Variants of Diaphanous 1 (DIAPH1).

One of the earliest mapped human deafness genes, DIAPH1, encodes the formin DIAPH1. To date, at least three distinct mutations in the C-terminal domains and two additional mutations in the N-terminal region are associated with autosomal dominant hearing loss. The underlying molecular mechanisms are not known, and the role of formins in the inner ear is not well understood. In this study, we use biochemical assays to test the hypotheses that autoinhibition and/or actin assembly activities are disrupted by DFNA1 mutations. Our results indicate that C-terminal mutant forms of DIAPH1 are functional in vitro and promote actin filament assembly. Similarly, N-terminal mutants are well-folded and have quaternary structures and thermal stabilities similar to those of the wild-type (WT) protein. The strength of the autoinhibitory interactions varies widely among mutants, with the ttaa, A265S, and I530S mutations having an affinity similar to that of WT and the 1213x and Δag mutations completely abolishing autoinhibition. These data indicate that, in some cases, hearing loss may be linked to weakened inhibition of actin assembly.

Investigation of the prevalence and catalytic activity of rubredoxin-fused alkane monooxygenases (AlkBs).

Interest in understanding the environmental distribution of the alkane monooxygenase (AlkB) enzyme led to the identification of over 100 distinct alkane monooxygenase (AlkB) enzymes containing a covalently bound, or fused, rubredoxin. The rubredoxin-fused AlkB from Dietzia cinnamea was cloned as a full-length protein and as a truncated protein with the rubredoxin domain deleted. A point mutation (V91W) was introduced into the full-length protein, with the goal of assessing how steric bulk in the putative substrate channel might affect selectivity. Based on activity studies with alkane and alkene substrates, the rubredoxin-fused AlkB oxidizes a similar range of alkane substrates relative to its rubredoxin domain-deletion counterpart. Oxidation of terminal alkenes generated both an epoxide and a terminal aldehyde. The products of V91W-mutant-catalyzed oxidation of alkenes had a higher aldehyde-to-epoxide ratio than the products formed in the presence of the wild type protein. These results are consistent with this mutation causing a structural change impacting substrate positioning.

Spire stimulates nucleation by Cappuccino and binds both ends of actin filaments.

The actin nucleators Spire and Cappuccino synergize to promote actin assembly, but the mechanism of their synergy is controversial. Together these proteins promote the formation of actin meshes, which are conserved structures that regulate the establishment of oocyte polarity. Direct interaction between Spire and Cappuccino is required for oogenesis and for in vitro synergistic actin assembly. This synergy is proposed to be driven by elongation and the formation of a ternary complex at filament barbed ends, or by nucleation and interaction at filament pointed ends. To mimic the geometry of Spire and Cappuccino in vivo, we immobilized Spire on beads and added Cappuccino and actin. Barbed ends, protected by Cappuccino, grow away from the beads while pointed ends are retained, as expected for nucleation-driven synergy. We found that Spire is sufficient to bind barbed ends and retain pointed ends of actin filaments near beads and we identified Spire's barbed-end binding domain. Loss of barbed-end binding increases nucleation by Spire and synergy with Cappuccino in bulk pyrene assays and on beads. Importantly, genetic rescue by the loss-of-function mutant indicates that barbed-end binding is not necessary for oogenesis. Thus, increased nucleation is a critical element of synergy both in vitro and in vivo.

The neuron-specific formin Delphilin nucleates nonmuscle actin but does not enhance elongation.

The formin Delphilin binds the glutamate receptor, GluRδ2, in dendritic spines of Purkinje cells. Both proteins play a role in learning. To understand how Delphilin functions in neurons, we studied the actin assembly properties of this formin. Formins have a conserved formin homology 2 domain, which nucleates and associates with the fast-growing end of actin filaments, influencing filament growth together with the formin homology 1 (FH1) domain. The strength of nucleation and elongation varies widely across formins. Additionally, most formins have conserved domains that regulate actin assembly through an intramolecular interaction. Delphilin is distinct from other formins in several ways: its expression is limited to Purkinje cells, it lacks classical autoinhibitory domains, and its FH1 domain has minimal proline-rich sequence. We found that Delphilin is an actin nucleator that does not accelerate elongation, although it binds to the barbed end of filaments. In addition, Delphilin exhibits a preference for actin isoforms, nucleating nonmuscle actin but not muscle actin, which has not been described or systematically studied in other formins. Finally, Delphilin is the first formin studied that is not regulated by intramolecular interactions. We speculate how the activity we observe is consistent with its localization in the small dendritic spines.

Actin filament assembly by bacterial factors VopL/F: Which end is up?

Competing models have been proposed for actin filament nucleation by the bacterial proteins VopL/F. In this issue, Burke et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201608104) use direct observation to demonstrate that VopL/F bind the barbed and pointed ends of actin filaments but only nucleate new filaments from the pointed end.

Structure of a putative ClpS N-end rule adaptor protein from the malaria pathogen Plasmodium falciparum.

The N-end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP-dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N-end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N-degrons. However, while the N-degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal-ClpS binds and discriminates peptides mimicking bona fide N-end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal-ClpS localizes to this plastid-like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti-malarial drugs aimed at disrupting parasite-specific protein quality control pathways.

Metavinculin Tunes the Flexibility and the Architecture of Vinculin-Induced Bundles of Actin Filaments.

Vinculin is an abundant protein found at cell-cell and cell-extracellular matrix junctions. In muscles, a longer splice isoform of vinculin, metavinculin, is also expressed. The metavinculin-specific insert is part of the C-terminal tail domain, the actin-binding site of both isoforms. Mutations in the metavinculin-specific insert are linked to heart disease such as dilated cardiomyopathies. Vinculin tail domain (VT) both binds and bundles actin filaments. Metavinculin tail domain (MVT) binds actin filaments in a similar orientation but does not bundle filaments. Recently, MVT was reported to sever actin filaments. In this work, we asked how MVT influences F-actin alone or in combination with VT. Cosedimentation and limited proteolysis experiments indicated a similar actin binding affinity and mode for both VT and MVT. In real-time total internal reflection fluorescence microscopy experiments, MVT's severing activity was negligible. Instead, we found that MVT binding caused a 2-fold reduction in F-actin's bending persistence length and increased susceptibility to breakage. Using mutagenesis and site-directed labeling with fluorescence probes, we determined that MVT alters actin interprotomer contacts and dynamics, which presumably reflect the observed changes in bending persistence length. Finally, we found that MVT decreases the density and thickness of actin filament bundles generated by VT. Altogether, our data suggest that MVT alters actin filament flexibility and tunes filament organization in the presence of VT. Both of these activities are potentially important for muscle cell function. Perhaps MVT allows the load of muscle contraction to act as a signal to reorganize actin filaments.

The role of formin tails in actin nucleation, processive elongation, and filament bundling.

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.

Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly.

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.

Autoinhibition of the formin Cappuccino in the absence of canonical autoinhibitory domains.

Formins are a conserved family of proteins known to enhance actin polymerization. Most formins are regulated by an intramolecular interaction. The Drosophila formin, Cappuccino (Capu), was believed to be an exception. Capu does not contain conserved autoinhibitory domains and can be regulated by a second protein, Spire. We report here that Capu is, in fact, autoinhibited. The N-terminal half of Capu (Capu-NT) potently inhibits nucleation and binding to the barbed end of elongating filaments by the C-terminal half of Capu (Capu-CT). Hydrodynamic analysis indicates that Capu-NT is a dimer, similar to the N-termini of other formins. These data, combined with those from circular dichroism, suggest, however, that it is structurally distinct from previously described formin inhibitory domains. Finally, we find that Capu-NT binds to a site within Capu-CT that overlaps with the Spire-binding site, the Capu-tail. We propose models for the interaction between Spire and Capu in light of the fact that Capu can be regulated by autoinhibition.

Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes.

Three semi-rational approaches, combinatorial site-saturation mutagenesis (CSSM) using a reduced amino acid set and two libraries based on C(orbit) and CRAM computational design algorithms targeting up to 10 active site residues, were used to engineer cytochrome P450 BM3 to demethylate dimethyl ether and hydroxylate propane and ethane. These small libraries (343-1028 variants) were all enriched with respect to the fraction functional and maximal activities compared with a random mutagenesis library and individual site-saturation libraries targeting the same residues. Despite high average amino acid substitution levels of 2.6, 5 and 7.5, the CSSM, C(orbit) and CRAM libraries had at least 75% of library members properly folded. Propane- and ethane-hydroxylating P450 BM3 variants were identified using all three mutagenesis approaches, with as few as two amino acid substitutions. The library designed using the CRAM algorithm, which sought to reduce the size of the binding pocket, produced both a higher number of active variants and variants supporting the greatest number of catalytic turnovers. The most active variant E32 supports 16 800 propane turnovers at 36% coupling, which rivals the activity of variants obtained after 10-12 rounds of directed evolution using random and site-saturation mutagenesis. None of the variants in this study achieved the complete re-specialization for propane hydroxylation (including 93% coupling) previously obtained via multiple rounds of mutagenesis and screening. However, these semi-rational approaches allowed for large jumps in sequence space to variants with the desired functions.

Structure and function of the interacting domains of Spire and Fmn-family formins.

Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

An improved pairwise decomposable finite-difference Poisson-Boltzmann method for computational protein design.

Our goal is to develop accurate electrostatic models that can be implemented in current computational protein design protocols. To this end, we improve upon a previously reported pairwise decomposable, finite difference Poisson-Boltzmann (FDPB) model for protein design (Marshall et al., Protein Sci 2005, 14, 1293). The improvement involves placing generic sidechains at positions with unknown amino acid identity and explicitly capturing two-body perturbations to the dielectric environment. We compare the original and improved FDPB methods to standard FDPB calculations in which the dielectric environment is completely determined by protein atoms. The generic sidechain approach yields a two to threefold increase in accuracy per residue or residue pair over the original pairwise FDPB implementation, with no additional computational cost. Distance dependent dielectric and solvent-exclusion models were also compared with standard FDPB energies. The accuracy of the new pairwise FDPB method is shown to be superior to these models, even after reparameterization of the solvent-exclusion model.

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function.

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.

Electrostatics in computational protein design.

Catalytic activity and protein-protein recognition have proven to be significant challenges for computational protein design. Electrostatic interactions are crucial for these and other protein functions, and therefore accurate modeling of electrostatics is necessary for successfully advancing protein design into the realm of protein function. This review focuses on recent progress in modeling electrostatic interactions in computational protein design, with particular emphasis on continuum models.