YjbH-enhanced proteolysis of Spx by ClpXP in Bacillus subtilis is inhibited by the small protein YirB (YuzO)

The Spx protein of Bacillus subtilis is a global regulator of the oxidative stress response. Spx concentration is controlled at the level of proteolysis by the ATP-dependent protease ClpXP and a substrate-binding protein, YjbH, which interacts with Spx. A yeast two-hybrid screen was carried out using yjbH as bait to uncover additional substrates or regulators of YjbH activity. Of the several genes identified in the screen, one encoded a small protein, YirB (YuzO), which elevated Spx concentration and activity in vivo when overproduced from an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible yirB construct. Pulldown experiments using extracts of B. subtilis cells producing a His-tagged YirB showed that native YjbH interacts with YirB in B. subtilis. Pulldown experiments using affinity-tagged Spx showed that YirB inhibited YjbH interaction with Spx. In vitro, YjbH-mediated proteolysis of Spx by ClpXP was inhibited by YirB. The activity of YirB is similar to that of the antiadaptor proteins that were previously shown to reduce proteolysis of a specific ClpXP substrate by interacting with a substrate-binding protein.     

Deletion of the carboxyl‐terminal residue disrupts the amino‐terminal folding,self‐association,and thermal stability of an amphipathic antimicrobial peptide

Understanding the complex relationship between amino acid sequence and protein behaviors, such as folding and self‐association, is a major goal of protein research. In the present work, we examined the effects of deleting a C‐terminal residue on the intrinsic properties of an amphapathic α‐helix of mastoparan‐B (MP‐B), an antimicrobial peptide with the sequence LKLKSIVSWAKKVL‐NH2. We used circular dichroism and nuclear magnetic resonance to demonstrate that the peptide MP‐B^[1‐13] displayed significant unwinding at the N‐terminal helix compared with the parent peptide of MP‐B, as the temperature increased when the residue at position 14 was deleted. Pulsed‐field gradient nuclear magnetic resonance data revealed that MP‐B forms a larger diffusion unit than MP‐B^[1‐13] at all experimental temperatures and continuously dissociates as the temperature increases. In contrast, the size of the diffusion unit of MP‐B^[1‐13] is almost independent of temperature. These findings suggest that deleting the flexible, hydrophobic amino acid from the C‐terminus of MP‐B is sufficient to change the intrinsic helical thermal stability and self‐association. This effect is most likely because of the modulation of enthalpic interactions and conformational freedom that are specified by this residue. Our results implicate terminal residues in the biological function of an antimicrobial peptide. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor‐binding specificities in different bacterial species

ClpXP, an AAA+ protease, plays key roles in protein‐quality control and many regulatory processes in bacteria. The N‐terminal domain of the ClpX component of ClpXP is involved in recognition of many protein substrates, either directly or by binding the SspB adaptor protein, which delivers specific classes of substrates for degradation. Despite very limited sequence homology between the E. coli and C. crescentus SspB orthologs, each of these adaptors can deliver substrates to the ClpXP enzyme from the other bacterial species. We show that the ClpX N domain recognizes different sequence determinants in the ClpX‐binding (XB) peptides of C. crescentus SspBα and E. coli SspB. The C. crescentus XB determinants span 10 residues and involve interactions with multiple side chains, whereas the E. coli XB determinants span half as many residues with only a few important side chain contacts. These results demonstrate that the N domain of ClpX functions as a highly versatile platform for peptide recognition, allowing the emergence during evolution of alternative adaptor‐binding specificities. Our results also reveal highly conserved residues in the XB peptides of both E. coli SspB and C. crescentus SspBα that play no detectable role in ClpX‐binding or substrate delivery.     

A flexible C‐terminal linker is required for proper FtsZ assembly in vitro and cytokinetic ring formation in vivo

Assembly of the cytoskeletal protein FtsZ into a ring‐like structure is required for bacterial cell division. Structurally, FtsZ consists of four domains: the globular N‐terminal core, a flexible linker, 8–9 conserved residues implicated in interactions with modulatory proteins, and a highly variable set of 4–10 residues at its very C terminus. Largely ignored and distinguished by lack of primary sequence conservation, the linker is presumed to be intrinsically disordered. Here we employ genetics, biochemistry and cytology to dissect the role of the linker in FtsZ function. Data from chimeric FtsZs substituting the native linker with sequences from unrelated FtsZs as well as a helical sequence from human beta‐catenin indicate that while variations in the primary sequence are well tolerated, an intrinsically disordered linker is essential for Bacillus subtilis FtsZ assembly. Linker lengths ranging from 25 to 100 residues supported FtsZ assembly, but replacing the B. subtilis FtsZ linker with a 249‐residue linker from Agrobacterium tumefaciens FtsZ interfered with cell division. Overall, our results support a model in which the linker acts as a flexible tether allowing FtsZ to associate with the membrane through a conserved C‐terminal domain while simultaneously interacting with itself and modulatory proteins in the cytoplasm.     

Influence of substituents on conformational preferences of helix foldamers of γ‐dipeptides

Conformational preferences of 9‐ and 14‐helix foldamers have been studied for γ‐dipeptides of 2‐aminocyclohexylacetic acid (γAc₆a) residues such as Ac‐(γAc₆a)₂‐NHMe ( 1 ), Ac‐(C^α‐Et‐γAc₆a)₂‐NHMe ( 2 ), Ac‐(γAc₆a)₂‐NHBn ( 3 ), and Ac‐(C^α‐Et‐γAc₆a)₂‐NHBn ( 4 ) at the M06‐2X/cc‐pVTZ//M06‐2X/6‐31 + G(d) level of theory to explore the influence of substituents on their conformational preferences. In the gas phase, the 9‐helix foldamer H9 and 14‐helix foldamer H14‐z are found to be most preferred for dipeptides 2 and 4 , respectively, as for dipeptides 1 and 3 , which indicates no remarkable influence of the C^α‐ethyl substitution on conformational preferences. The benzyl substitution at the C‐terminal end lead H14‐z to be the most preferred conformer for dipeptides 3 and 4 , whereas it is H9 for dipeptides 1 and 2 , which can be ascribed to the favored C? H···π interactions between the cyclohexyl group of the first residue and the C‐terminal benzyl group. There are only marginal changes in backbone structures and the distances and angles of H‐bonds for all local minima by C^α‐ethyl and/or benzyl substitutions. Although vibrational frequencies and intensities of the dipeptide 4 calculated at both M06‐2X/6‐31 + G(d) and M05‐2X/6‐31 + G(d) levels of theory are consistent with observed results in the gas phase, H14‐z is predicted to be most preferred by ΔG only at the former level of theory. Hydration did not bring the significant changes in backbone structures of helix foldamers for both dipeptide 1 and 4 . It is expected that the different substitutions at the C‐terminal end lead to the different helix foldamers, which may increase the resistance of helical structures to proteolysis and provide the more surface to the helical structures suitable for molecular recognition. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1077–1087, 2014.     

Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the alpha-helix have the same helical propensity

It was established previously that helical propensities of different amino acid residues in the middle of α‐helix in peptides and in proteins are very similar. The statistical analysis of the protein helices from the known three‐dimensional structures shows no difference in the frequency of noncharged residues in the middle and at the C terminus. Yet, experimental studies show distinctive differences for the helical propensities of noncharged residues in the middle and in the C terminus in model peptides. Is this a general effect, and is it applicable to protein helices or is it specific to the model alanine‐based peptides? To answer this question, the effects of substitutions at positions 28 (middle residue) and 32 (C2 position at the C terminus) of the α‐helix of ubiquitin on the stability of this protein are measured by using differential scanning calorimetry. The two data sets produce similar values for intrinsic helix propensity, leading to a conclusion that noncharged amino acid residues at the solvent‐exposed positions in the middle and at the C terminus of the α‐helix have the same helical propensity. This conclusion is further supported with an excellent correlation between the helix propensity scale obtained for the two positions in ubiquitin with the experimental helix propensity scale established previously and with the statistical distribution of the residues in protein helices.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural insights into the intramolecular interactions of centromere protein CENP‐I

In mitosis, the accurate segregation of sister chromosomes relies on kinetochore, a multiple subunits complex assembled on centromere of each sister chromosome. As a core component of inner kinetochore, CENP‐I plays important functions to mediate kinetochore assembly and supports the faithful chromosome segregation. The structures of the N‐terminus and C‐terminus of CENP‐I homologs in complex with CENP‐H/K have been reported, respectively. Unfortunately, the intramolecular interactions of CENP‐I are poorly understood, and how CENP‐I interacts with CENP‐M remains unknown. Here, we verified a unique helix α11, which forms the intramolecular interactions with N‐terminal HEAT repeats in fungal CENP‐I. Deletion of the helix α11 exposed the hydrophobic surface and resulted in the in vitro protein aggregation of N‐terminal HEAT repeats of fungal CENP‐I. The corresponding helix and its intramolecular interaction are highly conserved in human CENP‐I. Deletion of the corresponding helix in human CENP‐I dramatically reduced the functional activity to interact with CENP‐H and CENP‐M. Mutations of the conserved residues on the helix in human CENP‐I significantly weakened the binding to CENP‐M, but not CENP‐H, in HeLa cells. Therefore, our findings for the first time unveiled a conserved helix of CENP‐I, which is important for the intramolecular interaction and function, and would be helpful for understanding the structure basis of how CENP‐I mediates the kinetochore assembly during cell cycle and mitosis.     

Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii

Gas vesicles are proteinaceous, gas‐filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in‐silico 3D‐model of GvpA of the predicted coil‐α1‐β1‐β2‐α2‐coil structure is available and implies that the two β‐chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analyzed these variants in Haloferax volcanii ΔA + A_mut transformants for their ability to form gas vesicles (Vac⁺ phenotype). In most cases, an alanine substitution of a non‐polar residue did not abolish gas vesicle formation, but the replacement of single non‐polar by charged residues in β1 or β2 resulted in Vac^– transformants. A replacement of residues near the β‐turn altered the spindle‐shape to a cylindrical morphology of the gas vesicles. Vac^– transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt‐bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53 or Tyr54, led to Vac^– transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid‐state NMR.