SEA domain autoproteolysis accelerated by conformational strain: mechanistic aspects

A subclass of SEA (sea urchin sperm protein, enterokinase, and agrin) domain proteins undergoes autoproteolysis between glycine and serine in a conserved G^− 1S^+ 1VVV motif to generate stable heterodimers. Autoproteolysis has been suggested to involve only the intramolecular catalytic action of the conserved serine hydroxyl in combination with conformational strain of the glycine-serine peptide bond. We conducted a number of experiments and simulations on the SEA domain from the MUC1 mucin to test this mechanism. Alanine-scanning mutagenesis of polar residues in the vicinity of the cleavage site demonstrates that only the nucleophile at position + 1 is required for efficient proteolysis. Molecular modeling shows that an uncleaved trans peptide is incompatible with the native heterodimeric structure, resulting in disruption of secondary structure elements and distortion of the scissile peptide bond. Insertion of glycine residues (to obtain G_nG^− 1S^+ 1VVV motifs) appears to relieve strain, and autoproteolysis is 100 times slower in a 1G (n = 1) mutant and not measurable in 2G and 4G mutants. Removal of the catalytic serine hydroxyl hampers cleavage considerably, but measurable autoproteolysis of this S1098A mutant still proceeds in the presence of strain alone. The uncleaved SEA precursor populates interconverting partially folded conformations, and autoproteolysis coincides with adoption of proper β-sheet secondary structure and completed folding. Molecular dynamics simulations of the precursor show that the serine hydroxyl and the preceding glycine carbonyl carbon can be in van der Waals contact at the same time as the scissile peptide bond becomes strained. These observations are all consistent with autoproteolysis accelerated by N → O acyl shift and conformational strain imposed upon protein folding in a reaction for which the free-energy barrier is decreased by substrate destabilization rather than by transition-state stabilization. The energetics of this coupled folding and autoproteolysis mechanism is accounted for in an accompanying article.     

Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (T_m = 58 °C), enthalpy (ΔH_m = 50 ± 5 kcal mol^− 1), entropy (ΔS_m = 150 ± 10 cal mol^− 1 K^− 1), and average cooperative melting unit (〈n_c〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T_m = 40 °C; ΔH_m = 27 ± 2 kcal mol^− 1; ΔS_m = 86 ± 6 cal mol^− 1 K^− 1) and noncooperative unfolding (〈n_c〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.     

The Contribution of a Covalently Bound Cofactor to the Folding and Thermodynamic Stability of an Integral Membrane Protein

The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol^− 1 to 23.4 ± 1.5 kcal mol^− 1 at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol^− 1 towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol^− 1 to 10.9 ± 1.0 kcal mol^− 1, indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3-6 kcal mol^− 1. Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k_u^H2O, from ~1 × 10^− 16 s^− 1 to ~1 × 10^− 4 s^− 1 at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol^− 1 in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein-retinal interactions that increase the rigidity and compactness of the polypeptide chain.     

The energetic contribution of induced electrostatic asymmetry to DNA bending by a site-specific protein

DNA bending can be promoted by reducing the net negative electrostatic potential around phosphates on one face of the DNA, such that electrostatic repulsion among phosphates on the opposite face drives bending toward the less negative surface. To provide the first assessment of energetic contribution to DNA bending when electrostatic asymmetry is induced by a site-specific DNA binding protein, we manipulated the electrostatics in the EcoRV endonuclease-DNA complex by mutation of cationic side chains that contact DNA phosphates and/or by replacement of a selected phosphate in each strand with uncharged methylphosphonate. Reducing the net negative charge at two symmetrically located phosphates on the concave DNA face contributes − 2.3 kcal mol^− 1 to − 0.9 kcal mol^− 1 (depending on position) to complex formation. In contrast, reducing negative charge on the opposing convex face produces a penalty of + 1.3 kcal mol^− 1. Förster resonance energy transfer experiments show that the extent of axial DNA bending (about 50°) is little affected in modified complexes, implying that modification affects the energetic cost but not the extent of DNA bending. Kinetic studies show that the favorable effects of induced electrostatic asymmetry on equilibrium binding derive primarily from a reduced rate of complex dissociation, suggesting stabilization of the specific complex between protein and markedly bent DNA. A smaller increase in the association rate may suggest that the DNA in the initial encounter complex is mildly bent. The data imply that protein-induced electrostatic asymmetry makes a significant contribution to DNA bending but is not itself sufficient to drive full bending in the specific EcoRV-DNA complex.     

High-resolution structure of intramolecularly proteolyzed human mucin-1 SEA domain

SEA domains are ubiquitous in large proteins associated with highly glycosylated environments. Certain SEA domains undergo intramolecular proteolysis involving a nucleophilic attack of a serine hydroxyl group on the preceding glycine carbonyl. The mucin-1 (MUC1) SEA domain has been extensively investigated as a model of intramolecular proteolysis. Since neither a general base, a general acid, nor an oxyanion hole could be identified in MUC1 SEA, it has been suggested that proteolysis is accelerated by a non-planarity of the scissile peptide bond imposed by protein folding. A reactant distorted peptide bond has been also invoked to explain the autoproteolysis of several unrelated proteins. However, the only evidence of peptide distortion in MUC1 SEA stems from molecular dynamic simulations of the reactant modeled upon a single NMR structure of the cleaved product. We report the first high-resolution X-ray structure of cleaved MUC1 SEA. Structural comparison with uncleaved SEA domains suggests that the number of residues evolutionarily inserted in the cleaved loop of MUC1 SEA precludes the formation of a properly hydrogen-bonded beta turn. By sequence analysis, we show that this conformational frustration is shared by all known cleaved SEA domains. In addition, alternative conformations of the uncleaved precursor could be modeled in which the scissile peptide bond is planar. The implications of these structures for autoproteolysis are discussed in the light of the previous research on autoproteolysis.     

Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin

Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent ΔG°_UN(w) = − 3.1 ± 0.3 kcal mol^− 1; m_UN = − 1.7 kcal mol^− 1 M^− 1] and predominant α-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (β_I = 0.9 ± 0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.     

The Osmolyte TMAO Stabilizes Native RNA Tertiary Structures in the Absence of Mg: Evidence for a Large Barrier to Folding from Phosphate Dehydration

The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ∼ 0 for a hairpin secondary structure and between 0.70 and 1.85 kcal mol^− 1m^− 1 for four RNA tertiary structures (30-86 nt). Further analysis of two RNAs by small-angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg²⁺ and that the structures stabilized by TMAO and Mg²⁺ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg²⁺ ion chelation site formed in part by a buried phosphate, even though Mg²⁺ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy, + 211 cal mol^− 1m^− 1 for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66-173 water molecules in the RNA structures studied) and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg²⁺ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.     

Relative Domain Folding and Stability of a Membrane Transport Protein

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately + 2 kcal mol^− 1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of + 2 kcal mol^− 1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains.     

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.     

Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition

In this study, we have structurally characterized the amidase of a nitrile-degrading bacterium, Rhodococcus sp. N-771 (RhAmidase). RhAmidase belongs to amidase signature (AS) family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase. Recombinant RhAmidase exists as a dimer of about 107 kDa. RhAmidase can hydrolyze acetamide, propionamide, acrylamide and benzamide with kcat/Km values of 1.14 ± 0.23 mM^− 1s^− 1, 4.54 ± 0.09 mM^− 1s^− 1, 0.087 ± 0.02 mM^− 1s^− 1 and 153.5 ± 7.1 mM^− 1s^− 1, respectively. The crystal structures of RhAmidase and its inactive mutant complex with benzamide (S195A/benzamide) were determined at resolutions of 2.17 Å and 2.32 Å, respectively. RhAmidase has three domains: an N-terminal α-helical domain, a small domain and a large domain. The N-terminal α-helical domain is not found in other AS family enzymes. This domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. The large domain showed high structural similarities to those of other AS family enzymes. The Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket of RhAmidase is relatively narrow, due to the presence of the helix α13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases.     

Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu⁺ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔG_w° = 12.9 kJ mol^− 1, m = 4.1 kJ mol^− 1 M^− 1, C_m = 3 M, and ΔCp_w° = 0.93 kJ mol^− 1 K^− 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.     

Molecular rearrangement in POR macrodomains as a reason for the blue shift of chlorophyllide fluorescence observed after phototransformation

The activation energy and activation volume of the spectral blue shift subsequent to protochlorophyllide phototransformation (called Shibata shift in intact leaves) were studied in prolamellar body (PLB) and prothylakoid-(PT)-enriched membrane fractions prepared from dark-grown wheat (Triticum aestivum, L.) leaves. The measurements were done at 20, 30 and 40 °C and at various pressure values. The activation energy values were 181 ± 8 kJ mol^− 1 and 188 ± 6 kJ mol^− 1 for the PLBs and the PTs, respectively. The pressure stabilized the structure of the NADPH:protochlorophyllide oxidoreductase (POR) macrodomains; it prevented or slowed down the blue shift. There were no significant differences between the activation volumes of PLBs and PTs at 30 or 40 °C giving values around 100-125 ml mol^− 1 which correspond to changes in the tertiary structure of proteins but also resemble the volume changes occurring during the disaggregation of protein dimers or oligomers, or during dissociation of peripheral membrane proteins from membranes. The small differences in the activation parameters of PLBs and PTs indicate that molecular rearrangements inside the POR macrodomains are the primary reasons of the fluorescence blue shift; however, their lipid microenvironment must be also important in the initialization of the shift.     

Melting behavior and ligand binding of DNA intramolecular secondary structures

We use a variety of biophysical techniques to determine thermodynamic profiles, including hydration, for the unfolding of DNA stem-loop motifs (hairpin, a three-way junction and a pseudoknot) and their interaction with netropsin and random cationic copolymers. The unfolding thermodynamic data show that their helix-coil transition takes place according to their melting domains or sequences of their stems. All hairpins adopted the B-like conformation and their loop(s) contribute with an immobilization of structural water. The thermodynamic data of netropsin binding to the ^5′-AAATT-^3′/TTTAA site of each hairpin show affinities of ~ 10^6- 7 M^− 1, 1:1 stoichiometries, exothermic enthalpies of − 7 to − 12 kcal mol^− 1 (− 22 kcal mol^− 1 for the secondary site of the three-way junction), and water releases. Their interaction with random cationic copolymers yielded higher affinities of ~ 10⁶ M^− 1 with the more hydrophobic hairpins. This information should improve our current picture of how sequence and loops control the stability and melting behavior of nucleic acid molecules.     

The MUC1 SEA module is a self-cleaving domain

MUC1, a glycoprotein overexpressed by a variety of human adenocarcinomas, is a type I transmembrane protein (MUC1/TM) that soon after its synthesis undergoes proteolytic cleavage in its extracellular domain. This cleavage generates two subunits, alpha and beta, that specifically recognize each other and bind together in a strong noncovalent interaction. Proteolysis occurs within the SEA module, a 120-amino acid domain that is highly conserved in a number of heavily glycosylated mucin-like proteins. Post-translational cleavage of the SEA module occurs at a site similar to that in MUC1 in the glycoproteins IgHepta and MUC3. However, as in the case of other proteins containing the cleaved SEA module, the mechanism of MUC1 proteolysis has not been elucidated. Alternative splicing generates two transmembrane MUC1 isoforms, designated MUC1/Y and MUC1/X. We demonstrated here that MUC1/X, whose extracellular domain is comprised solely of the SEA module in addition to 30 MUC1 N-terminal amino acids, undergoes proteolytic cleavage at the same site as the MUC1/TM protein. In contrast, the MUC1/Y isoform, composed of an N-terminally truncated SEA module, is not cleaved. Cysteine or threonine mutations of the MUC1/X serine residue (Ser-63) immediately C-terminal to the cleavage site generated cleaved proteins, whereas mutation of the Ser-63 residue of MUC1/X to any other of 17 amino acids did not result in cleavage. In vitro incubation of highly purified precursor MUC1/X protein resulted in self-cleavage. Furthermore, addition of hydroxylamine, a strong nucleophile, markedly enhanced cleavage. Both these features are signature characteristics of self-cleaving proteins, and we concluded that MUC1 undergoes autoproteolysis mediated by an N --> O-acyl rearrangement at the cleavage site followed by hydrolytic resolution of the unstable ester and concomitant cleavage. It is likely that all cleaved SEA module-containing proteins follow a similar route.     

Structure, folding and stability of FimA, the main structural subunit of type 1 pili from uropathogenic Escherichia coli strains

Filamentous type 1 pili are responsible for attachment of uropathogenic Escherichia coli strains to host cells. They consist of a linear tip fibrillum and a helical rod formed by up to 3000 copies of the main structural pilus subunit FimA. The subunits in the pilus interact via donor strand complementation, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit. Here, we show that folding of FimA occurs at an extremely slow rate (half-life: 1.6 h) and is catalyzed more than 400-fold by the pilus chaperone FimC. Moreover, FimA is capable of intramolecular self-complementation via its own donor strand, as evidenced by the loss of folding competence upon donor strand deletion. Folded FimA is an assembly-incompetent monomer of low thermodynamic stability (− 10.1 kJ mol^− 1) that can be rescued for pilus assembly at 37 °C because FimC selectively pulls the fraction of unfolded FimA molecules from the FimA folding equilibrium and allows FimA refolding on its surface. Elongation of FimA at the C-terminus by its own donor strand generated a self-complemented variant (FimAa) with alternative folding possibilities that spontaneously adopts the more stable conformation (− 85.0 kJ mol^− 1) in which the C-terminal donor strand is inserted in the opposite orientation relative to that in FimA. The solved NMR structure of FimAa revealed extensive β-sheet hydrogen bonding between the FimA pilin domain and the C-terminal donor strand and provides the basis for reconstruction of an atomic model of the pilus rod.     

Structural Bases for Stability-Function Tradeoffs in Antibiotic Resistance

Preorganization of enzyme active sites for substrate recognition typically comes at a cost to the stability of the folded form of the protein; consequently, enzymes can be dramatically stabilized by substitutions that attenuate the size and preorganization “strain” of the active site. How this stability-activity tradeoff constrains enzyme evolution has remained less certain, and it is unclear whether one should expect major stability insults as enzymes mutate towards new activities or how these new activities manifest structurally. These questions are both germane and easy to study in β-lactamases, which are evolving on the timescale of years to confer resistance to an ever-broader spectrum of β-lactam antibiotics. To explore whether stability is a substantial constraint on this antibiotic resistance evolution, we investigated extended-spectrum mutants of class C β-lactamases, which had evolved new activity versus third-generation cephalosporins. Five mutant enzymes had between 100-fold and 200-fold increased activity against the antibiotic cefotaxime in enzyme assays, and the mutant enzymes all lost thermodynamic stability (from 1.7 kcal mol^− 1 to 4.1 kcal mol^− 1), consistent with the stability-function hypothesis. Intriguingly, several of the substitutions were 10-20 Å from the catalytic serine; the question of how they conferred extended-spectrum activity arose. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by X-ray crystallography. Distinct mechanisms of action, including changes in the flexibility and ground-state structures of the enzyme, are revealed for each mutant. These results explain the structural bases for the antibiotic resistance conferred by these substitutions and their corresponding decrease in protein stability, which will constrain the evolution of new antibiotic resistance.     

Thermodynamic analysis of hydration in human serum heme-albumin

Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 °C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S = ⁵/₂ manifold. Values of correlation times for tensor fluctuation (τ_v) and chemical exchange of water molecules (τ_M) show the expected temperature dependence, with activation enthalpies of −1.94 and −2.46 ± 0.2 kJ mol⁻¹, respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with ΔH = 68 ± 28 kJ mol⁻¹ and ΔS = 200 ± 80 J mol⁻¹ K⁻¹. These results highlight the role of the water solvent in heme-HSA structure-function relationships.     

Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.