Site-directed Chemical Cross-linking Research Articles

Determining Protein Structures by Iterating between Computation and Experiment

An increasing variety of experimental techniques for protein structure determination rely on the site-specific incorporation of probes. Examples include NMR using the paramagnetic relaxation enhancement (PRE) effect, fluorescence using FRET and related techniques, and site-directed chemical cross-linking quantified by mass-spectrometry. In each case, the probe locations must be carefully chosen, which poses a major challenge, as some probe locations are highly informative, whereas others yield little useful information. In the absence of structural knowledge, how does one efficiently choose probe locations? We present a method called Rational Probe Mutagenesis (RPM) that relies on iteration between computation and experiment to rapidly determine protein structures. RPM is based on the idea of reducing the “width” of the conformational ensemble as rapidly as possible. An ensemble is generated using whatever data is currently available. This ensemble is then analyzed to find the experiment that maximally discriminates between possible structures. The resulting experimental data is then used to generate a new updated ensemble and the process repeated. We demonstrate RPM on several simple model systems and a challenging real-world solid-state NMR dataset, where we show that RPM can choose highly informative experiments, leading to potential time and cost savings by avoiding redundant or uninformative experiments.

Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination.

The site-specific recombinase encoded by bacteriophage λ [λ Integrase (Int)] is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. In contrast to the other well-studied and highly exploited tyrosine recombinase family members, such as Cre and Flp, Int carries out a reaction that is highly directional, tightly regulated, and depends on an ensemble of accessory DNA bending proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental signals. Int recombinase is a heterobivalent DNA binding protein that binds via its small amino-terminal domain to high affinity arm-type DNA sites and via its large, compound carboxyl-terminal domain to core-type DNA sites, where DNA cleavage and ligation are executed. Each of the four Int protomers, within a multiprotein 400-kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site. Despite a wealth of genetic, biochemical, and functional information generated by many laboratories over the last 50 y, it has not been possible to decipher the patterns of Int bridges, an essential step in understanding the architectures responsible for regulated directionality of recombination. We used site-directed chemical cross-linking of Int in trapped Holliday junction recombination intermediates and recombination reactions with chimeric recombinases, to identify the unique and monogamous patterns of Int bridges for integrative and excisive recombination.

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

The maltose transporter MalFGK(2) is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction.

Modulation of E.Coli Dihydrofolate Reductase (DHFR) Activity by Site-Directed Chemical Cross-Linking (SDXL) of the M20 and FG Loops

Generally, proteins are comprised of highly complex structures and fluctuations of local or global character are believed to play critical roles in protein function. In the case of E.Coli dihydrofolate reductase (DHFR), previous structural studies have already revealed some aspects of DHFR's conformational flexibility. Among the most prominent displays of flexibility are motions between loops M20 and FG. We have employed site-directed chemical cross-linking (SDXL) to modulate loop motions and by extension modulate activity, flexibility, stability and substrate affinity. Toward this end, we have utilized both computational and experimental approaches. Target residues were identified by average distance matrices and principle component analysis calculated through molecular dynamic (MD) simulations at various temperatures. Activity measurements for a mirrored pair of cross-linked residues resulted in reduced activity for shorter cross-links, while longer cross-links returned the activity to near unmodified protein levels. Mass spectrometry was used to determine the effects on stability against thermal and chaotropic denaturation by observing changes in the charge state envelope associated with protein unfolding. Hydrogen/deuterium exchange (HDX) combined with mass spectrometry was used to probe local conformational changes both proximal and distal to the cross-link site. Finally with the ability to observe both free and bound species, mass spectrometry was able to monitor changes in substrate affinity as correlated with cross-link length. With this work we have shown that SDXL is able to modulate enzymatic activity and stability by controlling motions associated with thermal fluctuations.

Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

BackgroundWe applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins.Methodology/ResultsPhotoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.ConclusionAnalysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.

A Comparative Electron Paramagnetic Resonance Study of the Nucleotide-Binding Domains’ Catalytic Cycle in the Assembled Maltose ATP-Binding Cassette Importer

We present a quantitative analysis of conformational changes of the nucleotide-binding subunits, MalK2, of the maltose ATP-binding cassette importer MalFGK2 during the transport cycle. Distance changes occurring between selected residues were monitored in the full transporter by site-directed spin-labeling electron paramagnetic resonance spectroscopy and site-directed chemical cross-linking. We considered S83C and A85C from the conserved Q-loop and V117C located on the outer surface of MalK. Additionally, two native cysteines (C350, C360) were included in the study. On ATP binding, small rearrangements between the native sites, and no distance changes between positions 117 were detected. In contrast, positions 85 come closer together in the ATP-bound state and in the vanadate-trapped intermediate and move back toward the apo-state after ATP hydrolysis. The distance between positions 83 is shown to slightly decrease on ATP binding, and to further decrease after ATP hydrolysis. Results from cross-linking experiments are in agreement with these findings. The data are compared with in silico spin-labeled x-ray structures from both isolated MalK2 and the MalFGK2-E complex. Our results are consistent with a slightly modified “tweezers-like” model of closure and reopening of MalK2 during the catalytic cycle, and show an unforeseen potential interaction between MalK and the transmembrane subunit MalG.

Functional and Structural Interactions of the Transmembrane Domain X of NhaA, Na+/H+ Antiporter of Escherichia coli, at Physiological pH

The 3D structure of Escherichia coli NhaA, determined at pH 4, provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 7.0-8.5), many questions pertaining to the active state of NhaA have remained open, including the physiological role of helix X. Using a structural-based evolutionary approach in silico, we identified a segment of most conserved residues in the middle of helix X. These residues were then used as targets for functional studies at physiological pH. Cysteine-scanning mutagenesis showed that Gly303, in the middle of the conserved segment, is an essential residue and Cys replacement of Lys300 retains only Li+/H+ antiporter activity, with a 20-fold increase in the apparent KM for Li+. Cys replacements of Leu296 and Gly299 increase the apparent KM of the Na+/H+ antiporter for both Na+ and Li+. Accessibility test to N-ethylmaleimide and 2-sulfonatoethyl methanethiosulfonate showed that G299C, K300C, and G303C are accessible to the cytoplasm. Suppressor mutations and site-directed chemical cross-linking identified a functional and/or structural interaction between helix X (G295C) and helix IVp (A130C). While these results were in accordance with the acid-locked crystal structure, surprisingly, conflicting data were also obtained; E78C of helix II cross-links very efficiently with several Cys replacements of helix X, and E78K/K300E is a suppressor mutation of K300E. These results reveal that, at alkaline pH, the distance between the conserved center of helix X and E78 of helix II is drastically decreased, implying a pH-induced conformational change of one or both helices.

ATP Modulates Subunit-Subunit Interactions in an ATP-binding Cassette Transporter (MalFGK2) Determined by Site-directed Chemical Cross-linking

The binding protein-dependent maltose transport system of enterobacteria (MalFGK(2)), a member of the ATP-binding cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and of two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. In addition, an extracellular (periplasmic) substrate-binding protein (MalE) is required for activity. Ligand translocation and ATP hydrolysis are dependent on a signaling mechanism originating from the binding protein and traveling through MalF/MalG. Thus, subunit-subunit interactions in the complex are crucial to the transport process but the chemical nature of residues involved is poorly understood. We have investigated the proximity of residues in a conserved sequence ("EAA" loop) of MalF and MalG to residues in a helical segment of the MalK subunits by means of site-directed chemical cross-linking. To this end, single cysteine residues were introduced into each subunit at several positions and the respective malF and malG alleles were individually co-expressed with each of the malK alleles. Membrane vesicles were prepared from those double mutants that contained a functional transporter in vivo and treated with Cu(1,10-phenanthroline)(2)SO(4) or bifunctional cross-linkers. The results suggest that residues Ala-85, Lys-106, Val-114, and Val-117 in the helical segment of MalK, to different extents, participate in constitution of asymmetric interaction sites with the EAA loops of MalF and MalG. Furthermore, both MalK monomers in the complex are in close contact to each other through Ala-85 and Lys-106. These interactions are strongly modulated by MgATP, indicating a structural rearrangement of the subunits during the transport cycle. These data are discussed with respect to current transport models.

Proximity of Periplasmic Loops in the Metal-Tetracycline/H+ Antiporter of Escherichia coli Observed on Site-directed Chemical Cross-linking

Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A. (1999) FEBS Lett. 457, 169-173). To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline. The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control. Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed. Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6. In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking. The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.

Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ.

The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII. In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one face of helix VIII. Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices. Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one another toward the cytoplasmic side of the membrane. Furthermore, helices V and VIII are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane.

Site-directed chemical cross-linking demonstrates that helix IV is close to helices VII and XI in the lactose permease.

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease, each containing a single-Cys residue, were coexpressed, and proximity was studied. Paired Cys residues in helices IV (positions 114, 116, 119, 122, 125, or 129) and VII (227, 231, 232, 234, 235, 238, 239, 242, 243, 245, or 246) or XI (350, 353, 354, 357, 361, or 364) were tested for cross-linking in the presence of two rigid homobifunctional thiol-specific cross-linkers, N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N,N'-p-phenylenedimaleimide (p-PDM; 10 A). Cys residues in the middle of helix IV (position 119 or 122) cross-link to Cys residues in the middle of helix VII (position 238, 239, 242, or 243). In contrast, no cross-linking is evident with paired Cys residues at either end of helix IV (position 114, 116, 125, or 129) or helix VII (position 227, 231, 232, 234, 235, 245, or 246). On the other hand, Cys residues in the cytoplasmic half of helix IV (position 125 or 129) cross-link with Cys residues in the cytoplasmic half of helix XI (position 350, 353, or 354), while paired Cys residues at the periplasmic ends of the two helices do not cross-link. The results indicate that helices IV and VII cross in a scissors-like manner with the cytoplasmic end of helix IV tilting toward helix XI.

Tilting of helix I and ligand-induced changes in the lactose permease determined by site-directed chemical cross-linking in situ.

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of lactose permease, each with a single Cys residue, were co-expressed, and cross-linking was studied. The proximity of paired Cys residues in helices I (positions 11, 14, 15, 18, 25, 28, 29, or 32) and VII (positions 227, 231, 232, 234, 235, 238, 239, 241, 242, 245, or 246) was studied by using homobifunctional thiol-specific chemical linkers of different lengths and chemical properties. The results demonstrate that Cys residues on one face of the periplasmic half of helix I (positions 32, 29, 28, or 25) cross-link to Cys residues on one face of the periplasmic half of helix VII (242 or 245). In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices I (positions 11, 14, 15, or 18) and VII (positions 227, 230, 231, 232, 234, 235, 238, or 239). The results indicate that helices I and VII are in close proximity only at their periplasmic halves. Ligand binding decreases cross-linking efficiency of the Cys pair 28/245 or 25/242 with N, N'-o-phenylenedimaleimide (rigid 6 A) and increases efficiency with N,N'-p-phenylenedimaleimide (rigid 10 A) or 1,6-bismaleimidohexane (flexible 16 A), indicating that the inter-thiol distance is about 6 A in the absence of ligand and that ligand binding increases the distance up to 10 A. The inter-thiol distance for Cys pairs 29/245 or 32/245 is less than 6 A in the absence of ligand, and in the presence of ligand, distance increases to between 6 and 10 A. Taken together, the results indicate that ligand binding induces a translational or scissors-like rigid body movement of helix I and/or VII at the periplasmic interface between the helices.

Transmembrane helix tilting and ligand-induced conformational changes in the lactose permease determined by site-directed chemical crosslinking in situ

The N-terminal six transmenbrane helices (N 6) and the C-terminal six transmembrane helices (C 6) of the lactose permease of Escherichia coli, each with a Cys residue, were co-expressed independently, and crosslinking was studied. Proximity of paired Cys residues in helices II (position 49, 52, 53, 56, 57, 60, 63 or 67) and VII (position 227, 230, 231, 234, 238, 241, 242 or 245) or XI (position 350, 353, 354, 357, 361 or 364) was examined by using two homobifunctional thiol-specific crosslinking agents of different lengths (6 or 10 Å). The results demonstrate that a Cys residue placed in the periplasmic half of helix II (position 49, 52, 53 or 57) crosslinks to Cys residues in the periplasmic half of helix VII (position 241, 242 or 245). In contrast, no crosslinking is evident with paired-Cys residues in the cytoplasmic halves of helices II (position 60, 63 or 67) and VII (position 227, 230, 231, 234 or 238). Remarkably, a Cys residue in the cytoplasmic half of helix II (position 60, 63 or 67) crosslinks with a Cys residue in the cytoplasmic half of helix XI (position 350, 353 or 354), while paired-Cys residues at positions in the periplasmic halves of the two helices do not crosslink. Therefore, helix II is tilted in such a manner that the periplasmic end is close to helix VII, and the cytoplasmic end is close to helix XI. Furthermore, ligand-binding alters the crosslinking efficiency of paired-Cys residues in helices II and VII or XI, indicating that both interfaces are conformationally active. The results are consistent with the conclusion that ligand-binding induces a scissors-like movement of helices II and VII that increases interhelical distance by 3 to 4 Å at the periplasmic ends and decreases the distance by 3 to 4 Å at the approximate middle of the two transmembrane helices.

Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking

N and C-terminal halves of lactose permease, each with a single-Cys residue, were co-expressed, and crosslinking was studied. Iodine or N, N′- o-phenylenedimaleimide ( o-PDM; rigid 6 Å), crosslinks Asn245→Cys (helix VII) and Ile52→Cys or Ser53→Cys (helix II). N, N′- p-phenylenedimaleimide ( p-PDM; rigid 10 Å) crosslinks the 245/53 Cys pair weakly, but does not crosslink 245/52, and 1,6- bis-maleimidohexane (BMH; flexible 16 Å) crosslinks both pairs less effectively than o-PDM. Thus, 245 is almost equidistant from 52 and 53 by up to about 6 Å. BMH or p-PDM crosslinks Gln242→Cys and Ser53→Cys, but o-PDM is ineffective, indicating that distance varies by up to 10 Å. Ligand binding increases crosslinking of 245/53 with p-PDM or BMH, has little effect with o-PDM and decreases iodine crosslinking. Similar effects are observed with 245/52. Ligand increases 242/53 crosslinking with p-PDM or BMH, but no crosslinking is observed with o-PDM. Therefore, ligand induces a translational or scissors-like displacement of the helices by 3-4 Å. Crosslinking 245/53 inhibits transport indicating that conformational flexibility is important for function.

Structure and function of 5S rRNA in the ribosome.

5S rRNA is a small RNA molecule that is a component of a ribosome from almost all living organisms. In this review, we discuss the biogenesis of 5S rRNA and its properties as an independent structural domain of a ribosome as well as the current concepts concerning the higher order structure of 5S rRNA in free state and in its complexes with ribosomal proteins and its folding in the ribosome. Special attention is paid to recent experimental approaches that have been useful in 5S rRNA studies. Our own data on topography of 5S rRNA in the ribosomes are discussed in detail. The hypothesis describing the possible functional role of 5S rRNA for ribosome functioning is discussed.