Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive

Biogenesis of mitochondrial iron-sulfur (Fe/S) cluster proteins requires the interaction of multiple proteins with the highly conserved 14-kDa scaffold protein Isu, on which clusters are built prior to their transfer to recipient proteins. For example, the assembly process requires the cysteine desulfurase Nfs1, which serves as the sulfur donor for cluster assembly. The transfer process requires Jac1, a J-protein Hsp70 cochaperone. We recently identified three residues on the surface of Jac1 that form a hydrophobic patch critical for interaction with Isu. The results of molecular modeling of the Isu1-Jac1 interaction, which was guided by these experimental data and structural/biophysical information available for bacterial homologs, predicted the importance of three hydrophobic residues forming a patch on the surface of Isu1 for interaction with Jac1. Using Isu variants having alterations in residues that form the hydrophobic patch on the surface of Isu, this prediction was experimentally validated by in vitro binding assays. In addition, Nfs1 was found to require the same hydrophobic residues of Isu for binding, as does Jac1, suggesting that Jac1 and Nfs1 binding is mutually exclusive. In support of this conclusion, Jac1 and Nfs1 compete for binding to Isu. Evolutionary analysis revealed that residues involved in these interactions are conserved and that they are critical residues for the biogenesis of Fe/S cluster protein in vivo. We propose that competition between Jac1 and Nfs1 for Isu binding plays an important role in transitioning the Fe/S cluster biogenesis machinery from the cluster assembly step to the Hsp70-mediated transfer of the Fe/S cluster to recipient proteins.     

Pseudomonas aeruginosa cytochrome C(551): probing the role of the hydrophobic patch in electron transfer

Cytochrome c(551) from Pseudomonas aeruginosa is a monomeric redox protein of 82 amino-acid residues, involved in dissimilative denitrification as the physiological electron donor of cd(1) nitrite reductase. The distribution of charged residues on the surface of c(551) is very anisotropic: one side is richer in acidic residues whereas the other shows a ring of positive side chains, mainly lysines, located at the border of an hydrophobic patch which surrounds the heme crevice. In order to map in cytochrome c(551) the surface involved in electron transfer, we have introduced specific mutations in three residues belonging to the hydrophobic patch, namely Val23-->Asp, Pro58-->Ala and Ile59-->Glu. The effect of these mutations was analyzed studying both the self-exchange rate and the electron-transfer activity towards P. aeruginosa cd(1) nitrite reductase, the physiological partner and P. aeruginosa azurin, a copper protein often used as a model redox partner in vitro. Our results show that introduction of a negative charge in the hydrophobic patch severely hampers both homonuclear and heteronuclear electron transfer.     

Mutational analysis of the flagellar rotor protein FliN: identification of surfaces important for flagellar assembly and switching

FliN is a component of the flagellar switch complex in many bacterial species. The crystal structure is known for most of FliN, and a targeted cross-linking study (K. Paul and D. F. Blair, J. Bacteriol. 188:2502-2511, 2006) showed that it is organized in ring-shaped tetramers at the bottom of the basal body C ring. FliN is essential for flagellar assembly and direction switching, but its precise functions have not been defined. Here, we identify functionally important regions on FliN by systematic mutagenesis. Nonconservative mutations were made at positions sampling the surface of the protein, and the effects on flagellar assembly and function were measured. Flagellar assembly was disrupted by mutations in a conserved hydrophobic patch centered on the dimer twofold axis or by mutations on the surface that forms the dimer-dimer interface in the tetramer. The assembly defect in hydrophobic-patch mutants was partially rescued by overexpression of the flagellar export proteins FliH and FliI, and coprecipitation assays demonstrated a binding interaction between FliN and FliH that was weakened by mutations in the hydrophobic patch. Thus, FliN might contribute to export by providing binding sites for FliH or FliH-containing complexes. The region around the hydrophobic patch is also important for switching; certain mutations in or near the patch caused a smooth-swimming chemotaxis defect that in most cases could be partially rescued by overexpression of the clockwise-signaling protein CheY. The results indicate that FliN is more closely involved in switching than has been supposed, possibly contributing to the binding site for CheY on the switch.     

Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore

Site-directed mutagenesis was used to map the ligand-binding surface of the type II transforming growth factor-beta receptor extracellular domain (TbetaRII-ECD). Two putative ligand-binding sites were probed, the first being a predicted hydrophobic patch, the second being the finger 1 surface loop. Nine residues were mutated in the context of full-length TbetaRII and the effect of these mutations on ligand-binding and receptor signaling was analyzed. Complementary information was obtained by examining 'natural' evolutionary TbetaRII mutations. Together, the results indicate that residues within the finger 1 region, but not the hydrophobic patch, of the TbetaRII-ECD are required for productive ligand-binding. We conclude that, surprisingly, the ECDs of TbetaRII and type II activin receptor utilize distinct interacting surfaces for binding their respective ligands.     

NMR analysis of the transient complex between membrane photosystem I and soluble cytochrome c6

A structural analysis of the surface areas of cytochrome c(6), responsible for the transient interaction with photosystem I, was performed by NMR transverse relaxation-optimized spectroscopy. The hemeprotein was titrated by adding increasing amounts of the chlorophyllic photosystem, and the NMR spectra of the free and bound protein were analyzed in a comparative way. The NMR signals of cytochrome c(6) residues located at the hydrophobic and electrostatic patches, which both surround the heme cleft, were specifically modified by binding. The backbones of internal residues close to the hydrophobic patch of cytochrome c(6) were also affected, a fact that is ascribed to the conformational changes taking place inside the hemeprotein when interacting with photosystem I. To the best of our knowledge, this is the first structural analysis by NMR spectroscopy of a transient complex between soluble and membrane proteins.     

p53 R175H hydrophobic patch and H‐bond reorganization observed by MD simulation

Molecular dynamics simulations probe the origins of aberrant functionality of R175H p53, which normally prevent tumorigenesis. This hotspot mutation exhibits loss of its essential zinc cofactor, aggregation, and activation of gain of function promoters, characteristics contributing to the loss of normal p53 activity. This study provided molecular level insight into the reorganization of the hydrogen bonding network and the formation of a hydrophobic patch on the surface of the protein. The hydrogen bonding network globally redistributes at the expense of the stability of the β‐sandwich structure, and surface residues reorganize to expose a 250 Ų hydrophobic patch of residues covering approximately 2% of the solvent accessible surface. These changes could both stabilize the protein in the conformation exposing the patch to solvent to mediate the reported aggregation, and cause a destabilization in the area associated with DNA binding residues to affect the specificity. The development of the patch prior to loss of zinc indicates that stabilizing the patch quickly may prevent zinc loss. Considerations for rational design of small molecule therapeutics in light of the structural insight has been discussed and it suggest the positive ring around the hydrophobic patch and conserved residues may constitute a druggable site. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 176–185, 2016.     

The role of hydrophobic and negatively charged surface patches of lipid-free apolipoprotein A-I in lipid binding and ABCA1-mediated cholesterol efflux

Recent models of lipid-free apolipoprotein A-I, including a cross-link/homology model and an X-ray crystal structure have identified two potential functionally relevant “patches” on the protein surface. The first is a hydrophobic surface patch composed of leucine residues 42, 44, 46, and 47 and the second a negatively charged patch composed of glutamic acid residues 179, 191, and 198. To determine if these domains play a functional role, these surface patches were disrupted by site-directed mutagenesis and the bacterially expressed mutants were compared with respect to their ability to bind lipid and stimulate ABCA1-mediated cholesterol efflux. It was found that neither patch plays a significant functional role in the ability of apoA-I to accept cholesterol in an ABCA1-dependent manner, but that the hydrophobic patch did affect the ability of apoA-I to clear DMPC liposomes. Interestingly, contrary to previous predictions, disruption of the hydrophobic surface patch enhanced the lipid binding ability of apoA-I. The hydrophobic surface patch may be important to the structural stability of lipid-free apoA-I or may be a necessary permissive structural element for lipid binding.     

Solution structure of a membrane-anchored ubiquitin-fold (MUB) protein from Homo sapiens

The protein Bc059385, whose solution structure is reported here, is the human representative of a recently identified family of membrane-anchored ubiquitin-fold (MUB) proteins. Analysis of their similarity to ubiquitin indicates that homologous amino acid residues in MUBs form a hydrophobic surface very similar to the recognition patch surrounding Ile-44 in ubiquitin. This suggests that MUBs may interact with proteins containing an alpha-helical motif similar to those of some ubiquitin binding domains. A disordered loop common to MUBs may also provide a second protein interaction site. From the available data, it is probable that this protein is prenylated and associated with the membrane. With <20% identity to ubiquitin, the MUB family further expands the sequence space that maps to the beta-grasp fold, and adds membrane localization to its list of functional roles.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

Mapping binding residues in the Plasmodium vivax domain that binds Duffy antigen during red cell invasion

Summary Plasmodium vivax depends on interaction with the Duffy antigen/receptor for chemokines (DARC) for invasion of human erythrocytes. The 140 kDa P. vivax Duffy-binding protein (PvDBP) mediates interaction with DARC. The receptor-binding domain of PvDBP maps to its N-terminal, cysteine-rich region, region II (PvRII), which contains approximately 300 amino acid residues including 12 conserved cysteines. Using surface plasmon resonance, we show that binding of PvRII to DARC is a high-affinity interaction with a binding constant (K(D)) of 8.7 nM. The minimal binding domain of PvRII has been previously mapped to a central 170-amino-acid stretch that includes cysteines 5-8. Here, we have used site-directed mutagenesis and quantitative binding assays to map amino acid residues within PvRII that make contact with DARC. Of the seven alanine replacement mutations that had an effect on binding, five were mutations in hydrophobic residues suggesting that hydrophobic interactions play a major role in the interaction of PvDBP with DARC. Genetic diversity studies have shown that six of the seven binding residues identified in PvRII are conserved in P. vivax field isolates, which provides support for their role in interaction with DARC.     

Comparison of the binding sites for high-potential iron-sulfur protein and cytochrome c on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center

A tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) of purple bacterium, Rubrivivax gelatinosus, interacts with two types of soluble electron donors, cytochromes c and high-potential iron-sulfur protein (HiPIP), at a binding domain in the vicinity of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll. To clarify the mechanism of the interaction, the domain around heme 1 was examined using site-directed mutants that changed the surface charge in the region within 20 A from the heme edge. In the case of the interaction with soluble cytochrome c, a strong dependence on the sign of the introduced charge was observed in all mutants: positive charge inhibited the reaction rate, whereas additional negative charge accelerated it. This confirmed the electrostatic nature of the binding. Interaction with HiPIP was inhibited by a limited number of mutations at the close vicinity of heme 1, and no acceleration was observed (the effects of some mutations were independent of the sign of the introduced charge). The acidic residues which were critically important for the binding of cytochrome c showed much less contribution to the binding of HiPIP. The binding site for HiPIP appears to be mostly formed by uncharged and hydrophobic residues, occupying a significantly smaller area than the cytochrome-c-binding site. It is proposed that the docking of HiPIP to the RC in Rvi. gelatinosus is primarily controlled by hydrophobic contacts between protein surfaces, thus differing from the electrostatic mode of the RC-cytochrome c interaction.     

The interaction between syntaxin 1A and cystic fibrosis transmembrane conductance regulator Cl- channels is mechanistically distinct from syntaxin 1A-SNARE interactions

Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and synaptic Ca(2+) channels in addition to participating in SNARE complex assembly and membrane fusion. We exploited the isoform-specific nature of the interaction between syntaxin 1A and CFTR to identify residues in the H3 domain of this SNARE (SNARE motif) that influence CFTR binding and regulation. Mutating isoform-specific residues that map to the surface of syntaxin 1A in the SNARE complex led to the identification of two sets of hydrophilic residues that are important for binding to and regulating CFTR channels or for binding to the syntaxin regulatory protein Munc-18a. None of these mutations affected syntaxin 1A binding to other SNAREs or the assembly and stability of SNARE complexes in vitro. Conversely, the syntaxin 1A-CFTR interaction was unaffected by mutating hydrophobic residues in the H3 domain that influence SNARE complex stability and Ca(2+) channel regulation. Thus, CFTR channel regulation by syntaxin 1A involves hydrophilic interactions that are mechanistically distinct from the hydrophobic interactions that mediate SNARE complex formation and Ca(2+) channel regulation by this t-SNARE.     

A structure-function study of the C2 domain of cytosolic phospholipase A2. Identification of essential calcium ligands and hydrophobic membrane binding residues

The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a beta-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-alpha (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins.     

Crystal structure of the human receptor activity-modifying protein 1 extracellular domain

Receptor activity-modifying protein (RAMP) 1 forms a heterodimer with calcitonin receptor-like receptor (CRLR) and regulates its transport to the cell surface. The CRLR.RAMP1 heterodimer functions as a specific receptor for calcitonin gene-related peptide (CGRP). Here, we report the crystal structure of the human RAMP1 extracellular domain. The RAMP1 structure is a three-helix bundle that is stabilized by three disulfide bonds. The RAMP1 residues important for cell-surface expression of the CRLR.RAMP1 heterodimer are clustered to form a hydrophobic patch on the molecular surface. The hydrophobic patch is located near the tryptophan residue essential for binding of the CGRP antagonist, BIBN4096BS. These results suggest that the hydrophobic patch participates in the interaction with CRLR and the formation of the ligand-binding pocket when it forms the CRLR.RAMP1 heterodimer.     

Crystal structure of the Melampsora lini effector AvrP reveals insights into a possible nuclear function and recognition by the flax disease resistance protein P

The effector protein AvrP is secreted by the flax rust fungal pathogen (Melampsora lini) and recognized specifically by the flax (Linum usitatissimum) P disease resistance protein, leading to effector‐triggered immunity. To investigate the biological function of this effector and the mechanisms of specific recognition by the P resistance protein, we determined the crystal structure of AvrP. The structure reveals an elongated zinc‐finger‐like structure with a novel interleaved zinc‐binding topology. The residues responsible for zinc binding are conserved in AvrP effector variants and mutations of these motifs result in a loss of P‐mediated recognition. The first zinc‐coordinating region of the structure displays a positively charged surface and shows some limited similarities to nucleic acid‐binding and chromatin‐associated proteins. We show that the majority of the AvrP protein accumulates in the plant nucleus when transiently expressed in Nicotiana benthamiana cells, suggesting a nuclear pathogenic function. Polymorphic residues in AvrP and its allelic variants map to the protein surface and could be associated with differences in recognition specificity. Several point mutations of residues on the non‐conserved surface patch result in a loss of recognition by P, suggesting that these residues are required for recognition.     

Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.     

Structure of the N-terminal Mlp1-binding domain of the Saccharomyces cerevisiae mRNA-binding protein, Nab2

Nuclear abundant poly(A) RNA-binding protein 2 (Nab2) is an essential yeast heterogeneous nuclear ribonucleoprotein that modulates both mRNA nuclear export and poly(A) tail length. The N-terminal domain of Nab2 (residues 1-97) mediates interactions with both the C-terminal globular domain of the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), and the mRNA export factor, Gfd1. The solution and crystal structures of the Nab2 N-terminal domain show a primarily helical fold that is analogous to the PWI fold found in several other RNA-binding proteins. In contrast to other PWI-containing proteins, we find no evidence that the Nab2 N-terminal domain binds to nucleic acids. Instead, this domain appears to mediate protein:protein interactions that facilitate the nuclear export of mRNA. The Nab2 N-terminal domain has a distinctive hydrophobic patch centered on Phe73, consistent with this region of the surface being a protein:protein interaction site. Engineered mutations within this hydrophobic patch attenuate the interaction with the Mlp1 C-terminal domain but do not alter the interaction with Gfd1, indicating that this patch forms a crucial component of the interface between Nab2 and Mlp1.     

Features critical for membrane binding revealed by DivIVA crystal structure

DivIVA is a conserved protein in Gram‐positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N‐terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled‐coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low‐resolution crystal structure of the C‐terminal (Ct) domain displays a curved tetramer made from two parallel coiled‐coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.     

Mutational analysis of the subgroup A avian sarcoma and leukosis virus putative fusion peptide domain

Short hydrophobic regions referred to as fusion peptide domains (FPDs) at or near the amino terminus of the membrane-anchoring subunit of viral glycoproteins are believed to insert into the host membrane during the initial stage of enveloped viral entry. Avian sarcoma and leukosis viruses (ASLV) are unusual among retroviruses in that the region in the envelope glycoprotein (EnvA) proposed to be the FPD is internal and contains a centrally located proline residue. To begin analyzing the function of this region of EnvA, 20 substitution mutations were introduced into the putative FPD. The mutant envelope glycoproteins were evaluated for effects on virion incorporation, receptor binding, and infection. Interestingly, most of the single-substitution mutations had little effect on any of these processes. In contrast, a bulky hydrophobic substitution for the central proline reduced viral titers 15-fold without affecting virion incorporation or receptor binding, whereas substitution of glycine for the proline had only a nominal effect on EnvA function. Similar to other viral FPDs, the putative ASLV FPD has been modeled as an amphipathic helix where most of the bulky hydrophobic residues form a patch on one face of the helix. A series of alanine insertion mutations designed to interrupt the hydrophobic patch on the helix had differential effects on infectivity, and the results of that analysis together with the results observed with the substitution mutations suggest no correlation between maintenance of the hydrophobic patch and glycoprotein function.     

A hydrophobic cluster at the surface of the human plasma phospholipid transfer protein is critical for activity on high density lipoproteins

The plasma phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family, together with the cholesteryl ester transfer protein, the lipopolysaccharide binding protein (LBP) and the bactericidal permeability increasing protein (BPI). In the present study, we used the crystallographic data available for BPI to build a three-dimensional model for PLTP. Multiple sequence alignment suggested that, in PLTP, a cluster of hydrophobic residues substitutes for a cluster of positively charged residues found on the surface of LBP and BPI, which is critical for interaction with lipopolysaccharides. According to the PLTP model, these hydrophobic residues are situated on an exposed hydrophobic patch at the N-terminal tip of the molecule. To assess the role of this hydrophobic cluster for the functional activity of PLTP, single point alanine mutants were engineered. Phospholipid transfer from liposomes to high density lipoprotein (HDL) by the W91A, F92A, and F93A PLTP mutants was drastically reduced, whereas their transfer activity toward very low density lipoprotein and low density lipoprotein did not change. The HDL size conversion activity of the mutants was reduced to the same extent as the PLTP transfer activity toward HDL. Based on these results, we propose that a functional solvent-exposed hydrophobic cluster in the PLTP molecule specifically contributes to the PLTP transfer activity on HDL substrates.