A NOESY-HSQC simulation program, SPIRIT

A program SPIRIT (Simulation Program considering Incomplete Recovery of z magnetization and INEPT Transfer efficiency) has been developed to simulate three-dimensional NOESY-HSQC spectra. This program takes into account (1) different transfer efficiency during INEPT and reverse INEPT durations due to differential relaxation rates and¹ J coupling constants; (2) the different effect of the sensitivity-enhancement scheme on CH, CH₂ and CH₃ systems; and (3) incomplete recovery of longitudinal magnetization between scans. The simulation program incorporates anisotropic tumbling mode for symmetric tops, and allows for differential external relaxation rates for protons. Some well-defined internal motions, such as the fast rotation of methyl groups, are taken into account. The simulation program also allows for input of multiple conformations and their relative populations to calculate the average relaxation matrix to account for slow internal motions. With the SPIRIT program, the sensitivity-enhanced NOESY-HSQC experiment can be used directly in the evaluation of the accuracy of structures, which can potentially be improved by direct refinement against the primary data. Abbreviations: NOESY, nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single quantum correlation; INEPT, insensitive nuclei enhanced by polarization transfer.     

CP-HISQC: a better version of HSQC experiment for intrinsically disordered proteins under physiological conditions

¹H–¹⁵N HSQC spectroscopy is a workhorse of protein NMR. However, under physiological conditions the quality of HSQC spectra tends to deteriorate due to fast solvent exchange. For globular proteins only a limited number of surface residues are affected, but in the case of intrinsically disordered proteins (IDPs) HSQC spectra are thoroughly degraded, suffering from both peak broadening and loss of intensity. To alleviate this problem, we make use of the following two concepts. (1) Proton-decoupled HSQC. Regular HSQC and its many variants record the evolution of multi-spin modes, 2N_xH_z or 2N_xH_x, in indirect dimension. Under the effect of fast solvent exchange these modes undergo rapid decay, which results in severe line-broadening. In contrast, proton-decoupled HSQC relies on N_x coherence which is essentially insensitive to the effects of solvent exchange. Moreover, for measurements involving IDPs at or near physiological temperature, N_x mode offers excellent relaxation properties, leading to very sharp resonances. (2) Cross-polarization ¹H-to-¹⁵N transfer. If CP element is designed such as to lock both ¹H^N and water magnetization, the following transfer is effected: $ {\text{H}}_{\text{x}}^{\text{water}} \to {\text{H}}_{\text{x}}^{\text{N}} \to {\text{N}}_{\text{x}} . $ Thus water magnetization is successfully exploited to boost the amount of signal. In addition, CP element suffers less loss from solvent exchange, conformational exchange, and dipolar relaxation compared to the more popular INEPT element. Combining these two concepts, we have implemented the experiment termed CP-HISQC (cross-polarization assisted heteronuclear in-phase single-quantum correlation). The pulse sequence has been designed such as to preserve water magnetization and therefore can be executed with reasonably short recycling delays. In the presence of fast solvent exchange, k_ex ~ 100 s^?1, CP-HISQC offers much better spectral resolution than conventional HSQC-type experiments. At the same time it offers up to twofold gain in sensitivity compared to plain proton-decoupled HSQC. The new sequence has been tested on the sample of drkN SH3 domain at pH 7.5, 30 °C. High-quality spectrum has been recorded in less than 1 h, containing resonances from both folded and unfolded species. High-quality spectra have also been obtained for arginine side-chain H^εN^ε groups in the sample of short peptide Sos. For Arg side chains, we have additionally implemented (HE)NE(CD)HD experiment. Using ¹³C-labeled sample of Sos, we have demonstrated that proton-to-nitrogen CP transfer remains highly efficient in the presence of solvent exchange as fast as k_ex = 620 s^?1. In contrast, INEPT transfer completely fails in this regime.     

Rubredoxin as a paramagnetic relaxation-inducing probe

The paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c₃ was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2¹ and M18¹. The rubredoxin binding surface in the complex with cytochrome c₃ was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c₃ are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in ¹HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes.     

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: An application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T₁ of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T₁ is longer, no modulation of the coupling between metal centers can be detected.     

Optimized co-solute paramagnetic relaxation enhancement for the rapid NMR analysis of a highly fibrillogenic peptide

Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T ₁ relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T ₁ values across the entire sequence of the intrinsically disordered protein α-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in ‘proton-less’ NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from α₁-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.     

Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria

Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features.     

Zinc-substituted Desulfovibrio gigas desulforedoxins: resolving subunit degeneracy with nonsymmetric pseudocontact shifts

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe-4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with (15)N or (15)N + (13)C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D (15)N TOCSY-HSQC and NOESY-HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the (13)C(alpha) shifts were obtained from an HNCA experiment. Comparison of (1)H-(15)N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter- and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues >/=8.5 A from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo-oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.     

Refocusing revisited: An optimized,gradient-enhanced refocused HSQC and its applications in 2D and 3D NMR and in deuterium exchange experiments

Summary 2D ¹⁵N-¹H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T₂ relaxation during the refocusing delays. However, we show here that, when high ¹⁵N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH₂ geminal peaks and their truncation artefacts at a modest cost in sensitivity.     

Solution structure for an Encephalitozoon cuniculi adrenodoxin‐like protein in the oxidized state

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria‐like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome‐like organelles in E. cuniculi, the 128‐residue, adrenodoxin‐like protein Ec‐Adx (UniProt ID Q8SV19) in the [2Fe‐2S] ferredoxin superfamily. Oxidized Ec‐Adx contains a mixed four‐strand β‐sheet, β2‐β1‐β4‐β3 (↓↑↑↓), loosely encircled by three α‐helices and two 3₁₀‐helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin‐like proteins except for the absence of a fifth anti‐parallel β‐strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the ¹H‐¹⁵N HSQC spectrum of Ec‐Adx. These missing residues are clustered primarily in two regions, G48‐V61 and L94‐L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe‐2S] cluster. Missing amide resonances in ¹H‐¹⁵N HSQC spectra are detrimental to NMR‐based solution structure calculations because ¹H‐¹H NOE restraints are absent (glass half‐empty) and this may account for the absent β‐strand (β5) and the position of α3 in oxidized Ec‐Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe‐2S] cluster in oxidized Ec‐Adx (glass half‐full).     

The Crystal Structure of the [NiFe] Hydrogenase from the Photosynthetic Bacterium Allochromatium vinosum: Characterization of the Oxidized Enzyme (Ni-A State)

The crystal structure of the membrane-associated [NiFe] hydrogenase from Allochromatium vinosum has been determined to 2.1 Å resolution. Electron paramagnetic resonance (EPR) and Fourier transform infrared spectroscopy on dissolved crystals showed that it is present in the Ni-A state (> 90%). The structure of the A. vinosum [NiFe] hydrogenase shows significant similarities with [NiFe] hydrogenase structures derived from Desulfovibrio species. The amino acid sequence identity is ∼ 50%. The bimetallic [NiFe] active site is located in the large subunit of the heterodimer and possesses three diatomic non-protein ligands coordinated to the Fe (two CN⁻ , one CO). Ni is bound to the protein backbone via four cysteine thiolates; two of them also bridge the two metals. One of the bridging cysteines (Cys64) exhibits a modified thiolate in part of the sample. A mono-oxo bridging ligand was assigned between the metal ions of the catalytic center. This is in contrast to a proposal for Desulfovibrio sp. hydrogenases that show a di-oxo species in this position for the Ni-A state. The additional metal site located in the large subunit appears to be a Mg²⁺ ion. Three iron-sulfur clusters were found in the small subunit that forms the electron transfer chain connecting the catalytic site with the molecular surface. The calculated anomalous Fourier map indicates a distorted proximal iron-sulfur cluster in part of the crystals. This altered proximal cluster is supposed to be paramagnetic and is exchange coupled to the Ni³⁺ ion and the medial [Fe₃S₄]⁺ cluster that are both EPR active (S = 1/2 species). This finding of a modified proximal cluster in the [NiFe] hydrogenase might explain the observation of split EPR signals that are occasionally detected in the oxidized state of membrane-bound [NiFe] hydrogenases as from A. vinosum.     

On the interpretation of electron paramagnetic resonance spectra of binuclear iron-sulfur proteins

A method is described for the interpretation of electron paramagnetic resonance spectra of reduced binuclear iron-sulfur proteins. The g_y values for any protein can be analyzed so that both the symmetry and the extent of covalency at the paramagnetic site can be parameterized. These parameters can be related to the chemical composition of the paramagnetic center, the protein-dependent charge delocalization of the unpaired electron, and the geometric arrangement at the reduced iron atom. These analyses may ultimately be used to rationalize certain aspects of the redox potentials of the various iron-sulfur proteins.     

Sequence-specific assignments of the 1H nuclear magnetic resonance spectra of reduced high-potential ferredoxin (HiPIP) from Chromatium vinosum.

The 1H resonances of the high-potential [4Fe-4S]2+ ferredoxin from Chromatium vinosum have been assigned through conventional sequential methodology applied to 2D NMR spectra. Almost 80% of the residues were identified using standard 2D COSY, HOHAHA, and NOESY pulse sequences. These residues correspond to four segments of the primary structure that do not interact strongly with the iron-sulfur cluster. A minor correction to the amino acid sequence is strongly suggested by these NMR data. Additional protons more sensitive to the proximity of the cluster were assigned by a combination of NOESY experiments with fast repetition rates and short mixing times and of HOHAHA spectra recorded with reduced spin-lock duration aimed at compensating for the short relaxation rates. Hence, the contributions of 79 residues out of 85 were identified in NMR spectra, among which the assignments of 64 residues were completed. Even the fastest relaxing protons, like those of the cysteine ligands, could be correlated, partly because the strong hyperfine shifts isolate them from the crowded diamagnetic region. However, other protons, in particular those involved in NH-S hydrogen bonds with the iron-sulfur cluster, were more difficult to identify, most probably because their relatively broad signals overlapped with those of protons not or less perturbed by the active site. The availability of the major part of the 1H NMR assignments has enabled the detection and identification of many interresidue NOESY cross peaks. These data are in full agreement with the elements of secondary structure previously revealed by X-ray crystallographic analysis of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.     

Paramagnetic relaxation enhancement‐assisted structural characterization of a partially disordered conformation of ubiquitin

Nuclear magnetic resonance (NMR) is a powerful tool to study three‐dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N₁ and N₂ and plus another folded conformation, I, in which some amide group signals of residues 33–41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well‐converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure‐induced loss of backbone amide group signals at residues 28, 33, 36, and 39–41 like the wild‐type, with a similar but smaller effect on CαH and CβH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N₂ conformation, although the amide groups at residues 33–41 have more heterogeneous conformations and more contact with water, which differ from the N₁ and N₂ states. The PRE‐assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.     

Conformation of pseudoazurin in the 152 kDa electron transfer complex with nitrite reductase determined by paramagnetic NMR

Copper-containing nitrite reductase is able to catalyze the reduction of nitrite with a turnover rate of several hundreds per second. Electrons for the reaction are donated by the electron transfer protein pseudoazurin. The process of protein complex formation, electron transfer and dissociation must occur on the millisecond timescale to enable the fast turnover of the enzyme. The structure of this transient protein complex has been studied using paramagnetic NMR spectroscopy. Gadolinium complexes were attached specifically through two engineered Cys residues on three sites on the surface of nitrite reductase, causing strong distance-dependent relaxation effects on the residues of pseudoazurin. Docking of the two proteins based on these NMR-derived distance restraints and the chemical shift perturbation data shows convergence to a cluster of structures with an average root-mean-square deviation of 1.5 Å. The binding interface consists of polar and non-polar residues surrounded by charges. The interprotein distance between the two type-1 copper sites is 15.5(± 0.5) Å, enabling fast interprotein electron transfer. The NMR-based lower limit estimate of 600 s⁻¹ for the dissociation rate constant and the fast electron transfer are consistent with the transient nature of the complex.     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis

Summary The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by ¹H NMR spectroscopy. TOCSY and NOESY experiments in H₂O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H₂O and D₂O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary structural elements identified in Tl Fd are a three-stranded antiparallel -strand involving the termini of the protein, a double -strand (also antiparallel), two -helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the diamagnetic protein matrix indicate that the overall folding pattern of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio gigas [Kissinger et al. (1991) J. Mol. Biol., 219, 693–723]. The influence of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.Abbreviations Fd ferredoxin - HiPiP high-potential iron-sulfur proteins - Dg Desulfovibrio gigas - Av Azotobacter vinelandii - Pf Pyrococcus furiosus - Tl Thermococcus litoralis - Pa Peptostreptococcus asaccharolyticus - Bt Bacillus thermoproteolyticus - Cp Clostridium pasteurianum - Ca Clostridium acidi urici - Da Desulfovibrio africanus - Tm Thermatoga maritima - NOE nuclear Overhauser effect - NOESY 2D NOE spectroscopy - MCOSY 2D magnitude correlation spectroscopy - TOCSY total correlation spectroscopy To whom correspondence should be addressed.     

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme–copper site in cytochrome bo 3, E. coli

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme–copper dinuclear site in Escherichia coli cytochrome bo _3, a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T ₁, of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T ₂, of the dinuclear coupled pair Fe(III)–Cu_B(II) in the oxidized form of QO that is too short to measure directly. In the case of the F′ state, the relaxation properties of the heme–copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu_B(II) coupled by approximately 1.5–3 cm⁻¹ to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine–histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.     

Negatively Charged Lipid Membranes Promote a Disorder-Order Transition in the Yersinia YscU Protein

The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscU_C) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscU_C variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscU_C that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.     

Negatively Charged Lipid Membranes Promote a Disorder-Order Transition in the Yersinia YscU Protein

The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscU_C) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscU_C variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscU_C that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.