David W. Keizer

DNA Binding: a Novel Function of Pseudomonas aeruginosa Type IV Pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.

Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili.

Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal α-helix. Engineering of a truncated pilin fromPseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an α-helix at the N terminus that lies across a 4-stranded antiparallel β-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.

Binding hot spot for invasion inhibitory molecules on Plasmodium falciparum apical membrane antigen 1

ABSTRACT Apical membrane antigen 1 (AMA1) is expressed in schizont-stage malaria parasites and sporozoites and is thought to be involved in the invasion of host red blood cells. AMA1 is an important vaccine candidate, as immunization with this antigen induces a protective immune response in rodent and monkey models of human malaria. Additionally, anti-AMA1 polyclonal and monoclonal antibodies inhibit parasite invasion in vitro. We have isolated a 20-residue peptide (R1) from a random peptide library that binds to native AMA1 as expressed by Plasmodium falciparum parasites. Binding of R1 peptide is dependent on AMA1 having the proper conformation, is strain specific, and results in the inhibition of merozoite invasion of host erythrocytes. The solution structure of R1, as determined by nuclear magnetic resonance spectroscopy, contains two structured regions, both involving turns, but the first region, encompassing residues 5 to 10, is hydrophobic and the second, at residues 13 to 17, is more polar. Several lines of evidence reveal that R1 targets a “hot spot” on the AMA1 surface that is also recognized by other peptides and monoclonal antibodies that have previously been shown to inhibit merozoite invasion. The functional consequence of binding to this region by a variety of molecules is the inhibition of merozoite invasion into host erythrocytes. The interaction between these peptides and AMA1 may further our understanding of the molecular mechanisms of invasion by identifying critical functional regions of AMA1 and aid in the development of novel antimalarial strategies.

Metal Binding Affinities of Arabidopsis Zinc and Copper Transporters: Selectivities Match the Relative, but Not the Absolute, Affinities of their Amino-Terminal Domains

HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P(1B)-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn(2+), and the third transports Cu(+). Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu(I) binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn(2+)-ATPases. The three MBDs HMA2n (residues 1-79), HMA4n (residues 1-96), and HMA7n (residues 56-127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn(II) and Cu(I) of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn(II) are higher than that of HMA7n by a factor of 20-30, but the relative affinities for Cu(I) are inverted by a factor of 30-50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu(I) with an affinity between those of HMA4n and HMA7n but binds Zn(II) more weakly than either parent protein does. The four MBDs bind Cu(I) more strongly than Zn(II) by factors of >10(6). It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu(+) over Zn(2+). This information highlights the potential toxicity of Cu(+) in vivo and why copper sensor proteins are approximately 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein-protein interactions), and compartmentalization. The structure of Zn(II)-bound HMA4n defined by NMR confirmed the predicted ferredoxin betaalphabetabetaalphabeta fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed (113)Cd chemical shift in [(113)Cd]HMA4n was consistent with a Cd(II)S(2)OX (X = O or N) coordination sphere. The Zn(II) form of the Cu(I) transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn(II)-HMA7n)(m)]. Each Zn(II) ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.

Binding site for the C-domain of insulin-like growth factor (IGF) binding protein-6 on IGF-II; implications for inhibition of IGF actions

Insulin‐like growth factors (IGFs) are important mediators of growth and IGF‐binding proteins (IGFBPs) 1–6 regulate IGF actions. As IGFBP C‐terminal domains contribute to high‐affinity IGF binding, we have defined the binding site for the C‐domain of IGFBP‐6 on IGF‐II using NMR. This site lies adjacent to and between the binding sites for the IGFBP N‐domain and IGF‐I receptor (IGFIR), which have previously been found on opposite sides of the IGF molecule. The C‐domain is therefore likely to interfere with IGF binding to the IGFIR, providing a structural basis for the potent inhibitory effects of intact IGFBPs on IGF actions.

Interactions between elicitins and radish Raphanus sativus

Abstract. Two responses to elicitins are described in cultivars of radish (Raphanus sativus L.). Type I, exhibited by the cultivar Daikon, is characterised by wilting and desiccation within 24 h of elicitin application and was previously reported as the sensitive response (S. Kamoun et al. 1993, Mol Plant-Microbe Interact 6: 15–25). At 1 μg elicitin · g−1 FW radish tissue, symptoms appeared after 8 h, a sensitivity comparable to that shown by tobacco to β elicitins (J.-C. Pernollet et al., 1993, Physiol Mol Plant Pathol 42: 53–67; S. Kamoun et al., 1993, Mol Plant-Microbe Interact 6: 15–25). Elicitin failed to induce these symptoms in the cultivar White Icicle, even at 100 μg · g−1 FW of tissue. However, a different response (Type II) with symptoms resembling senescence appeared in White Icicle after 48 h and were fully developed by 72 h. The Type II response was induced at levels of elicitin above 0.3 μg · g−1 FW. Elicitin-treated Daikon leaves held at 100% relative humidity, rather than ambient (50–60%) did not wilt and by 72 h displayed Type II symptoms. When treated Daikon leaves were removed to ambient humidity at any time during the latent period, they developed Type I symptoms within 2 h. Although Type I symptoms were suppressed in Daikon at high humidity, there was no indication that leaf diffusion resistance or plant water conductance were affected. Protoplasts from the cultivar Daikon responded to elicitin by H+ uptake and K+ release, with maximal response at 300 pM. The response was eliminated by K252a or staurosporine. Daikon protoplasts also showed transient uptake/secretion of Ca2+ on elicitin addition. Protoplasts from White Icicle gave neither of these responses. Both Daikon and White Icicle phenotypes could be transferred to progeny of Daikon-White Icicle crosses and in the F2 generation three phenotypes, including a null, segregated. Only those F2 plants which exhibited the Daikon phenotype produced protoplasts which responded to elicitin.

Measuring translational diffusion coefficients of peptides and proteins by PFG‑NMR using band‑selective RF pulses

Abstract Molecular translational self-diffusion, a measure of diffusive motion, provides information on the effective molecular hydrodynamic radius, as well as information on the properties of media or solution through which the molecule diffuses. Protein translational diffusion measured by pulsed-field gradient nuclear magnetic resonance (PFG-NMR) has seen increased application in structure and interaction studies, as structural changes or protein–protein interactions are often accompanied by alteration of their effective hydrodynamic radii. Unlike the analysis of complex mixtures by PFG-NMR, for monitoring changes of protein translational diffusion under various conditions, such as different stages of folding/unfolding, a partial region of the spectrum or even a single resonance is sufficient. We report translational diffusion coefficients measured by PFG-NMR with a modified stimulated echo (STE) sequence where band-selective pulses are employed for all three 1H RF pulses. Compared with conventional non-selective sequence, e.g. the BPP-LED sequence, the advantage of this modified band-selective excitation short transient (BEST) version of STE (BEST-STE) sequence is multi-fold, namely: (1) potential sensitivity gain as in generalized BEST-based sequences, (2) water suppression is no longer required as the magnetization of solvent water is not perturbed during the measurement, and (3) dynamic range problems due to the presence of intense resonances from molecules other than the protein or peptide of interest, such as non-deuterated detergent micelles, are avoided.

Mimotopes of Apical Membrane Antigen 1: Structures of Phage-Derived Peptides Recognized by the Inhibitory Monoclonal Antibody 4G2dc1 and Design of a More Active Analogue

ABSTRACT Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum is an integral membrane protein that plays a key role in merozoite invasion of host erythrocytes. A monoclonal antibody, 4G2dc1, recognizes correctly folded AMA1 and blocks merozoite invasion. Phage display was used to identify peptides that bind to 4G2dc1 and mimic an important epitope of AMA1. Three of the highest-affinity binders—J1, J3, and J7—were chosen for antigenicity and immunogenicity studies. J1 and J7 were found to be true antigen mimics since both peptides generated inhibitory antibodies in rabbits (J. L. Casey et al., Infect. Immun. 72:1126-1134, 2004). In the present study, the solution structures of all three mimotopes were investigated by nuclear magnetic resonance spectroscopy. J1 adopted a well-defined region of structure, which can be attributed in part to the interactions of Trp11 with surrounding residues. In contrast, J3 and J7 did not adopt an ordered conformation over the majority of residues, although they share a region of local structure across their consensus sequence. Since J1 was the most structured of the peptides, it provided a template for the design of a constrained analogue, J1cc, which shares a structure similar to that of J1 and has a disulfide-stabilized conformation around the Trp11 region. J1cc binds with greater affinity to 4G2dc1 than does J1. These peptide structures provide the foundation for a better understanding of the complex conformational nature of inhibitory epitopes on AMA1. With its greater conformational stability and higher affinity for AMA1, J1cc may be a better in vitro correlate of immunity than the peptides identified by phage display.

C-Terminal Domain of Insulin-like Growth Factor (IGF) Binding Protein 6: Conformational Exchange and Its Correlation with IGF-II Binding †

Insulin-like growth factor binding proteins (IGFBPs) function as carriers and regulators of the insulin-like growth factors (IGF-I and -II). Within the family of six binding proteins, IGFBP-6 is unique in having a 20-100-fold higher affinity for IGF-II over IGF-I and appears to act primarily as an inhibitor of IGF-II actions. We have recently determined the solution structure of the C-terminal domain of IGFBP-6 (C-BP-6), which shows the presence of substantial flexible regions, including three loop regions. In this paper, we report results from (15)N relaxation measurements carried out in both the laboratory and rotating frames. Analysis of conventional (15)N relaxation data (R(1), R(2), and steady-state (15)N-[(1)H] nuclear Overhauser effect) indicated that there was a considerable number of residues involved in conformational/chemical exchange. Measurements of off-resonance (15)N R(1)(rho) in the rotating frame and (15)N relaxation dispersion using an in- and antiphase coherence-averaged Carr-Purcell-Meiboom-Gill sequence were thus carried out to gain further insight into the solution dynamics of C-BP-6. Although the off-resonance (15)N relaxation data showed no clear evidence for residues undergoing microsecond motion, the (15)N relaxation dispersion data allowed us to identify 15 residues that clearly exhibit submilli- to millisecond motion. A good correlation was observed between residues exhibiting motion at submilli- to millisecond time scales and those affected by IGF-II binding, as identified through the perturbation of nuclear magnetic resonance (NMR) spectra of C-BP-6 following IGF-II addition. A complete NMR relaxation study of C-BP-6 dynamics in complex with IGF-II was hampered by peak broadening and disappearance of C-BP-6 in the presence of IGF-II. Nonetheless, current results strongly suggest possible conformation switching or population shifting between pre-existing conformations in C-BP-6 upon binding to IGF-II.

The BECN1 N-terminal domain is intrinsically disordered

ABSTRACT BECN1/Beclin 1 has a critical role in the early stages of autophagosome formation. Recently, structures of its central and C-terminal domains were reported, however, little structural information is available on the N-terminal domain, comprising a third of the protein. This lack of structural information largely stems from the inability to produce this region in a purified form. Here, we describe the expression and purification of the N-terminal domain of BECN1 (residues 1 to 150) and detailed biophysical characterization, including NMR spectroscopy. Combined, our studies demonstrated at the atomic level that the BECN1 N-terminal domain is intrinsically disordered, and apart from the BH3 subdomain, remains disordered following interaction with a binding partner, BCL2L1/BCL-XL. In addition, the BH3 domain α-helix induced upon interaction with BCL2L1 reverts to a disordered state when the complex is dissociated by exposure to a competitive inhibitor. No significant interactions between N- and C-terminal domains were detected.