Low frequency vibrational modes in proteins: Changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers

As a step toward understanding their functional role, the low frequency vibrational motions (<300 cm−1) that are coupled to optical excitation of the primary donor bacteriochlorophyll cofactors in the reaction center from Rhodobacter sphaeroides were investigated. The pattern of hydrogen-bonding interaction between these bacteriochlorophylls and the surrounding protein was altered in several ways by mutation of single amino acids. The spectrum of low frequency vibrational modes identified by femtosecond coherence spectroscopy varied strongly between the different reaction center complexes, including between different mutants where the pattern of hydrogen bonds was the same. It is argued that these variations are primarily due to changes in the nature of the individual modes, rather than to changes in the charge distribution in the electronic states involved in the optical excitation. Pronounced effects of point mutations on the low frequency vibrational modes active in a protein-cofactor system have not been reported previously. The changes in frequency observed indicate a strong involvement of the protein in these nuclear motions and demonstrate that the protein matrix can increase or decrease the fluctuations of the cofactor along specific directions.

Non-enzymatic and enzymatic hydrolysis of alkyl halides: A haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an SN2 displacement reaction

The semiempirical PM3 method, calibrated against ab initio HF/6–31+G(d) theory, has been used to elucidate the reaction of 1,2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO− with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

Raman spectral evidence for hydration forces between collagen triple helices

Hydration forces are thought to result from the energetic cost of water rearrangement near macromolecular surfaces. Raman spectra, collected on the same collagen samples on which these forces were measured, reveal a continuous change in water hydrogen-bonding structure as a function of separation between collagen triple helices. The varying spectral parameters track the force-distance curve. The energetic cost of water “restructuring,” estimated from the spectra, is consistent with the measured energy of intermolecular interaction. These correlations support the idea that the change in water structure underlies the exponentially varying forces seen in this system at least over the 13–18-Å range of interaxial separations.

Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

The extended environment of mononuclear metal centers in protein structures

The objectives of this and the following paper are to identify commonalities and disparities of the extended environment of mononuclear metal sites centering on Cu, Fe, Mn, and Zn. The extended environment of a metal site within a protein embodies at least three layers: the metal core, the ligand group, and the second shell, which is defined here to consist of all residues distant less than 3.5 Å from some ligand of the metal core. The ligands and second-shell residues can be characterized in terms of polarity, hydrophobicity, secondary structures, solvent accessibility, hydrogen-bonding interactions, and membership in statistically significant residue clusters of different kinds. Findings include the following: (i) Both histidine ligands of type I copper ions exclusively attach the Nδ1 nitrogen of the histidine imidazole ring to the metal, whereas histidine ligands for all mononuclear iron ions and nearly all type II copper ions are ligated via the Nɛ2 nitrogen. By contrast, multinuclear copper centers are coordinated predominantly by histidine Nɛ2, whereas diiron histidine contacts are predominantly Nδ1. Explanations in terms of steric differences between Nδ1 and Nɛ2 are considered. (ii) Except for blue copper (type I), the second-shell composition favors polar residues. (iii) For blue copper, the second shell generally contains multiple methionine residues, which are elements of a statistically significant histidine–cysteine–methionine cluster. Almost half of the second shell of blue copper consists of solvent-accessible residues, putatively facilitating electron transfer. (iv) Mononuclear copper atoms are never found with acidic carboxylate ligands, whereas single Mn2+ ion ligands are predominantly acidic and the second shell tends to be mostly buried. (v) The extended environment of mononuclear Fe sites often is associated with histidine–tyrosine or histidine–acidic clusters.

Classification of mononuclear zinc metal sites in protein structures

Our study of the extended metal environment, particularly of the second shell, focuses in this paper on zinc sites. Key findings include: (i) The second shell of mononuclear zinc centers is generally more polar than hydrophobic and prominently features charged residues engaged in an abundance of hydrogen bonding with histidine ligands. Histidine–acidic or histidine–tyrosine clusters commonly overlap the environment of zinc ions. (ii) Histidine tautomeric metal bonding patterns in ligating zinc ions are mixed. For example, carboxypeptidase A, thermolysin, and sonic hedgehog possess the same ligand group (two histidines, one unibidentate acidic ligand, and a bound water), but their histidine tautomeric geometries markedly differ such that the carboxypeptidase A makes only Nδ1 contacts, thermolysin makes only Nɛ2 contacts, and sonic hedgehog uses one of each. Thus the presence of a similar ligand cohort does not necessarily imply the same topology or function at the active site. (iii) Two close histidine ligands HXmH, m ≤ 5, rarely both coordinate a single metal ion in the Nδ1 tautomeric conformation, presumably to avoid steric conflicts. Mononuclear zinc sites can be classified into six types depending on the ligand composition and geometry. Implications of the results are discussed in terms of divergent and convergent evolution.

The anticoagulant activation of antithrombin by heparin

Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-Å structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin–protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.

(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure–activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N′-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1′ enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1′/S2′ subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA

Nondistorting C4′ backbone adducts serve as molecular tools to analyze the strategy by which a limited number of human nucleotide excision repair (NER) factors recognize an infinite variety of DNA lesions. We have constructed composite DNA substrates containing a noncomplementary site adjacent to a nondistorting C4′ adduct to show that the loss of hydrogen bonding contacts between partner strands is an essential signal for the recruitment of NER enzymes. This specific conformational requirement for excision is mediated by the affinity of xeroderma pigmentosum group A (XPA) protein for nonhybridizing sites in duplex DNA. XPA recognizes defective Watson–Crick base pair conformations even in the absence of DNA adducts or other covalent modifications, apparently through detection of hydrophobic base components that are abnormally exposed to the double helical surface. This recognition function of XPA is enhanced by replication protein A (RPA) such that, in combination, XPA and RPA constitute a potent molecular sensor of denatured base pairs. Our results indicate that the XPA–RPA complex may promote damage recognition by monitoring Watson–Crick base pair integrity, thereby recruiting the human NER system preferentially to sites where hybridization between complementary strands is weakened or entirely disrupted.

Tight Asp-85–Thr-89 association during the pump switch of bacteriorhodopsin

Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O—H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-18O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O—D group of Thr-89 in D2O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O—H (O—D) group of Thr-89 in M is indicated by a shift in the C⩵O stretching vibration of the former because of 18O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.

Protein–RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.

Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7,8-dihydro-8-oxo-2′-deoxyguanosine:2′-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1–Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.

Topological testing of the mechanism of homology search promoted by RecA protein

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA–single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA–ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA–ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA–ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson–Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA–ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.

Polar residues drive association of polyleucine transmembrane helices

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161–166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154–160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-Å resolution

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-Å resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an ɛ-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.