Titus M. Franzmann

Some like it hot: the structure and function of small heat-shock proteins.

Small heat-shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. Recent evidence suggests that they maintain protein homeostasis by binding proteins in non-native conformations, thereby preventing substrate aggregation. Some members of the sHsp family are inactive or only partially active under physiological conditions, and transition toward the active state is induced by specific triggers, such as elevated temperature. Release of substrate proteins bound to sHsps requires cooperation with ATP-dependent chaperones, suggesting that sHsps create a reservoir of non-native proteins for subsequent refolding.

The eye lens chaperone α-crystallin forms defined globular assemblies

α-Crystallins are molecular chaperones that protect vertebrate eye lens proteins from detrimental protein aggregation. αB-Crystallin, 1 of the 2 α-crystallin isoforms, is also associated with myopathies and neuropathological diseases. Despite the importance of α-crystallins in protein homeostasis, only little is known about their quaternary structures because of their seemingly polydisperse nature. Here, we analyzed the structures of recombinant α-crystallins using biophysical methods. In contrast to previous reports, we show that αB-crystallin assembles into defined oligomers consisting of 24 subunits. The 3-dimensional (3D) reconstruction of αB-crystallin by electron microscopy reveals a sphere-like structure with large openings to the interior of the protein. αA-Crystallin forms, in addition to complexes of 24 subunits, also smaller oligomers and large clusters consisting of individual oligomers. This propensity might explain the previously reported polydisperse nature of α-crystallin.

Activation of the Chaperone Hsp26 Is Controlled by the Rearrangement of Its Thermosensor Domain

Cells respond to a sudden increase in temperature with the transcription of a special set of genes, a phenomenon known as the heat shock response. In the yeast S. cerevisiae, the molecular chaperone Hsp26 is one component of the heat shock response. Hsp26 has the remarkable ability to sense increases in temperature directly and can switch from an inactive to a chaperone-active state. The underlying principle of this temperature regulation has remained enigmatic. Hsp26 variants with altered spectroscopic properties allowed us to identify structural elements controlling this activation process. We show that temperature sensing by Hsp26 is a feature of its middle domain that changes its conformation within a narrow temperature range. This structural rearrangement allows Hsp26 to respond autonomously and directly to heat stress by reversibly unleashing its chaperone activity. Thus, the Hsp26 middle domain is a thermosensor and intrinsic regulator of chaperone activity.

Protein refolding by pH-triggered chaperone binding and release.

Molecular chaperones are typically either adenosine triphosphate (ATP) dependent or rely heavily on their ATP-dependent chaperone counterparts in order to promote protein folding. This presents a challenge to chaperones that are localized to ATP-deficient cellular compartments. Here we describe a mechanism by which the pH-regulated acid stress chaperone HdeA is capable of independently facilitating the refolding of acid-denatured proteins in the bacterial periplasm, which lacks both ATP and ATP-dependent chaperone machines. Our results are consistent with a model in which HdeA stably binds substrates at low pH, thereby preventing their irreversible aggregation. pH neutralization subsequently triggers the slow release of substrate proteins from HdeA, keeping the concentration of aggregation-sensitive intermediates below the threshold where they begin to aggregate. This provides a straightforward and ATP-independent mechanism that allows HdeA to facilitate protein refolding. Unlike previously characterized chaperones, HdeA appears to facilitate protein folding by using a single substrate binding-release cycle. This cycle is entirely regulated by the external environment and is therefore energy-neutral for the bacteria.

Identification of a Hypochlorite-specific Transcription Factor from Escherichia coli

Background: Hypochlorite is strongly bactericidal and used as disinfectant; yet, a response regulator allowing adaptation to the inflicted stress is so far unknown. Results: The transcription factor YjiE specifically confers hypochlorite resistance and is an atypical dodecameric regulator that undergoes DNA-induced dissociation to dimers and tetramers. Conclusion: YjiE protects cells from hypochlorite-induced oxidative damage by triggering a specific stress response. Significance: This is the first described hypochlorite-specific regulator. Hypochlorite is a powerful oxidant produced by neutrophils to kill invading microorganisms. Despite this important physiological role of HOCl in fighting bacterial infections, no hypochlorite-specific stress response has been identified yet. Here, we identified a hypochlorite-responsive transcription factor, YjiE, which is conserved in proteobacteria and eukaryotes. YjiE forms unusual dodecameric ring-like structures in vitro that undergo large DNA-induced conformational changes to form dimers and tetramers as shown by transmission electron microscopy and analytical ultracentrifugation. Such smaller oligomers are predominant in hypochlorite-stressed cells and are the active species as shown by fluorescence anisotropy and analytical ultracentrifugation. YjiE regulates a large number of genes upon hypochlorite stress. Among them are genes involved in cysteine, methionine biosynthesis, and sulfur metabolism (up-regulated) and genes involved in iron acquisition and homeostasis (down-regulated), thus supposedly replenishing oxidized metabolites and decreasing the hypochlorite-mediated amplification of intracellular reactive oxygen species. As a result, YjiE specifically confers hypochlorite resistance to E. coli cells. Thus, to our knowledge, YjiE is the first described hypochlorite-specific transcription factor.

Regulatory Circuits of the AAA+ Disaggregase Hsp104

Yeast Hsp104 is an AAA+ chaperone that rescues proteins from the aggregated state. Six protomers associate to form the functional hexamer. Each protomer contains two AAA+ modules, NBD1 and NBD2. Hsp104 converts energy provided by ATP into mechanical force used to thread polypeptides through its axial channel, thereby disrupting protein aggregates. But how the action of its 12 AAA+ domains is co-ordinated to catalyze disaggregation remained unexplained. Here, we identify a sophisticated allosteric network consisting of three distinct pathways that senses the nucleotide state of AAA+ modules and transmits this information across the Hsp104 hexamer. As a result of this communication, NBD1 and NBD2 each adopt two distinct conformations (relaxed and tense) that are reciprocally regulated. The key element in the network is the NBD1-ATP state that enables Hsp104 to switch from a barely active [RT] state to a highly active [TR] state. This concerted switch involves both cis and trans protomer interactions and provides Hsp104 with the mechanistic scaffold to catalyze disaggregation. It prepares the chaperone for polypeptide binding and activates NBD2 to generate the power strokes required to resolve protein aggregates. ATP hydrolysis in NBD1 resolves the high affinity [TR] state and switches the chaperone back into the low affinity [RT] state. Our model integrates previously unexplained observations and provides the first comprehensive map of nucleotide-related allosteric signals in a class-1 AAA+ protein.

Tandem Acyl Carrier Proteins in the Curacin Biosynthetic Pathway Promote Consecutive Multienzyme Reactions with a Synergistic Effect

Modular polyketide synthases (PKSs) are large multifunctional biosynthetic enzyme systems that assemble a remarkable array of secondary metabolites with a broad spectrum of biological activities. They are typically comprised of a chain initiation and termination module and multiple chain elongation modules. Chain elongation intermediates are covalently attached to a flexible phosphopantetheine (Ppant) arm of acyl carrier protein (ACP) domains embedded in the PKS modules. They are readily combined with other biosynthetic elements (e.g., non-ribosomal peptide synthetases (NRPSs) and 3-hydroxy-3-methylglutaryl (HMG) b-branching enzyme cassettes) for metabolic diversification. These additional enzymes can alter the assembly and tailoring schemes of the original PKS pathways and result in unexpected structural modifications. Many of these hybrid systems remain poorly understood, and biochemical studies have yet to reveal how new features are incorporated and refined in the pathways. However, knowledge gained from these versatile assemblies will guide future engineering of novel secondary metabolic systems. The biosynthetic pathway for curacin A (Figure 1a), a mixed-polyketide nonribosomal-peptide marine natural product with potent anticancer activities, is a prominent example of a hybrid PKS/NRPS system. In the curacin A chain initiation module, a bifunctional decarboxylase/ S-acetyltransferase (GNATL) domain, instead of a “canonical” acyltransferase (AT) domain, catalyzes chain initiation. A sulfotransferase domain inserted in the chain termination module was found to catalyze decarboxylative chain termination to form an unusual terminal olefin in the final product. In addition, a 10-enzyme assembly, including a halogenase (Hal) domain, a HMG enzyme cassette, and an enoyl reductase (ER) domain, is incorporated into the first two PKS chain elongation modules, and catalyzes formation of a cyclopropane moiety (Figure 1a). One key component in the curacin A HMG enzyme cassette is a tandem ACP3 (ACPI–ACPII–ACPIII) tridomain with three almost identical ACPs. All of the other enzymes in the 10-enzyme assembly, except CurB ACPIV and CurC ketosynthase (KS), catalyze modifications only on substrates linked to the tandem ACP3. Moreover, the ACP3 carries substrates for the first chain elongation module and is proposed to mediate substrate transfer to a peptidyl carrier protein (PCP) domain in a downstream CurF NRPS module, instead of an inactive CurF PKS module (Figure 1a). Therefore, the tandem ACP3 might be involved in complex and sequential protein interactions with at least nine enzymes, including CurA AT, KS and Hal domains, CurD HMG-ACP synthase (HCS), CurE dehydratase (ECH1), and CurF decarboxylase (ECH2), ER, condensation/cyclization (Cy) and PCP domains (Figure 1). Our current understanding of tandem ACP domains in polyketide or fatty acid pathways is that they enable sequential substrate modifications to occur in parallel. Genetic studies based on in vivo assays of truncations and active site mutants of tandem ACP domains in the mupirocin and polyunsaturated fatty acid pathways suggested that they can improve product yields. 10] Biochemical studies on a tandem di-ACP in the bacillaene pathway showed that each domain can function in parallel. Interestingly, tandem trior di-ACP domains are frequently associated with the HMG enzyme cassettes in hybrid PKS pathways (e.g., jamaicamide, bacillaene, mupriocin, and pederin), which catalyze consecutive modifications on tandem ACP-linked substrates (see the Supporting Information, Figure S1). Sequence comparison of the curacin A and jamaicamide pathways shows that these three ACP domains are the most conserved components in the 10-enzyme assembly (ca. 95% sequence identity, except for linker regions between ACPs). Thus, the ACP3 might serve as a special “relay station” to support [*] Dr. L. Gu, E. B. Eisman, Prof. Dr. D. H. Sherman Life Sciences Institute, Departments of Medicinal Chemistry, Chemistry, and Microbiology and Immunology University of Michigan, Ann Arbor, MI 48109 (USA) E-mail: davidhs@umich.edu

Characterization of a highly flexible self-assembling protein system designed to form nanocages.

The design of proteins that self‐assemble into well‐defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two‐component protein system, designated A‐(+) and A‐(−), in which self‐assembly is mediated by complementary electrostatic interactions between two coiled‐coil sequences appended to the C‐terminus of a homotrimeric enzyme with C3 symmetry. The coiled‐coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self‐assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo‐electron microscopy (EM). We show that upon mixing, A‐(+) and A‐(−) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo‐EM of size‐fractionated material shows that A‐(+) and A‐(−) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.

The Crystal Structure of Escherichia coli Group 4 Capsule Protein GfcC Reveals a Domain Organization Resembling That of Wza

We report the 1.9 Å resolution crystal structure of enteropathogenic Escherichia coli GfcC, a periplasmic protein encoded by the gfc operon, which is essential for assembly of group 4 polysaccharide capsule (O-antigen capsule). Presumed gene orthologs of gfcC are present in capsule-encoding regions of at least 29 genera of Gram-negative bacteria. GfcC, a member of the DUF1017 family, is comprised of tandem β-grasp (ubiquitin-like) domains (D2 and D3) and a carboxyl-terminal amphipathic helix, a domain arrangement reminiscent of that of Wza that forms an exit pore for group 1 capsule export. Unlike the membrane-spanning C-terminal helix from Wza, the GfcC C-terminal helix packs against D3. Previously unobserved in a β-grasp domain structure is a 48-residue helical hairpin insert in D2 that binds to D3, constraining its position and sequestering the carboxyl-terminal amphipathic helix. A centrally located and invariant Arg115 not only is essential for proper localization but also forms one of two mostly conserved pockets. Finally, we draw analogies between a GfcC protein fused to an outer membrane β-barrel pore in some species and fusion proteins necessary for secreting biofilm-forming exopolysaccharides.

Structural and functional analysis of the DEAF-1 and BS69 MYND domains.

DEAF-1 is an important transcriptional regulator that is required for embryonic development and is linked to clinical depression and suicidal behavior in humans. It comprises various structural domains, including a SAND domain that mediates DNA binding and a MYND domain, a cysteine-rich module organized in a Cys4-Cys2-His-Cys (C4-C2HC) tandem zinc binding motif. DEAF-1 transcription regulation activity is mediated through interactions with cofactors such as NCoR and SMRT. Despite the important biological role of the DEAF-1 protein, little is known regarding the structure and binding properties of its MYND domain. Here, we report the solution structure, dynamics and ligand binding of the human DEAF-1 MYND domain encompassing residues 501–544 determined by NMR spectroscopy. The structure adopts a ββα fold that exhibits tandem zinc-binding sites with a cross-brace topology, similar to the MYND domains in AML1/ETO and other proteins. We show that the DEAF-1 MYND domain binds to peptides derived from SMRT and NCoR corepressors. The binding surface mapped by NMR titrations is similar to the one previously reported for AML1/ETO. The ligand binding and molecular functions of the related BS69 MYND domain were studied based on a homology model and mutational analysis. Interestingly, the interaction between BS69 and its binding partners (viral and cellular proteins) seems to require distinct charged residues flanking the predicted MYND domain fold, suggesting a different binding mode. Our findings demonstrate that the MYND domain is a conserved zinc binding fold that plays important roles in transcriptional regulation by mediating distinct molecular interactions with viral and cellular proteins.