Effects of high pressure and temperature on the wild-type and F29W mutant forms of the N-domain of avian troponin C

The N-domain of troponin C (residues 1-90) regulates muscle contraction through conformational changes induced by Ca2+ binding. A mutant form of the isolated domain of avian troponin C (F29W) has been used in previous studies to observe conformational changes that occur upon Ca2+ binding, and pressure and temperature changes. Here we set out to determine whether the point mutation itself has any effects on the protein structure and its stability to pressure and temperature in the absence of Ca2+. Molecular dynamics simulations of the wild-type and mutant protein structures suggested that both structures are identical except in the main chain and the loop I region near the mutation site. Also, the simulations proposed that an additional cavity had been created in the core of the mutant protein. To determine whether such a cavity would affect the behavior of the protein when subjected to high pressures and temperatures, we performed 1H-NMR experiments at 300, 400, and 500 MHz on the wild-type and F29W mutant forms of the chicken N-domain troponin C in the absence of Ca2+. We found that the mutant protein at 5 kbar pressures had a destabilized beta-sheet between the Ca2+-binding loops, an altered environment near Phe-26, and reduced local motions of Phe-26 and Phe-75 in the core of the protein, probably due to a higher compressibility of the mutant. Under the same pressure conditions, the wild-type domain exhibited little change. Furthermore, the hydrophobic core of the mutant protein denatured at temperatures above 47 degrees C, while the wild-type was resistant to denaturation up to 56 degrees C. This suggests that the partially exposed surface mutation (F29W) significantly destabilizes the N-domain of troponin C by altering the packing and dynamics of the hydrophobic core.     

Comparison of the D-lactate stereospecific dehydrogenase of Limulus polyphemus with active-site regions of L-lactate dehydrogenases

Lactate dehydrogenase (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) from the horseshoe crab, Limulus polyphemus, a dimeric enzyme stereospecific for D-lactate, has been purified by affinity chromatography. Maleyl tryptic peptides containing arginine residues isolated from the Limulus enzyme have been characterized and sequenced. The small peptides obtained from similarly treated L-lactate-specific enzyme homologs define major portions of the substrate and coenzyme binding regions and are virtually identical among L-lactate-specific enzymes. Although the six small peptides and free arginine isolated from the Limulus enzyme indicate that the small number of arginine tryptic peptides are located in a few discrete consecutive clusters similarly to the L-lactate dehydrogenases, the peptides nevertheless show no obvious sequence homology to the corresponding peptides from L-lactate dehydrogenases. These results indicate that this lactate dehydrogenase of altered substrate specificity either evolved with major rearrangements of the active site if it evolved from an L-lactate dehydrogenase, or that D-lactate dehydrogenases have evolved from a different protein. The results contradict proposed models which suggest that minor changes in the spatial orientation of pyruvate resulting from minimal rearrangement of the active site could accommodate the change in substrate specificity.     

Effects of eglin-c binding to thermitase: three-dimensional structure comparison of native thermitase and thermitase eglin-c complexes.

Thermitase is a thermostable member of the subtilisin family of serine proteases. Four independently determined crystal structures of the enzyme are compared in this study: a high resolution native one and three medium resolution complexes of thermitase with eglin-c, grown from three different calcium concentrations. It appeared that the B-factors of the thermitase eglin complex obtained at 100 mM CaCl2 and elucidated at 2.0 A resolution are remarkably similar to those of the 1.4 A native structure: the main chain atoms have an rms difference of only 2.3 A2; for all atoms this difference is 4.6 A2. The rms positional differences between these two structures of thermitase are 0.31 A for the main chain atoms and 0.58 A for all atoms. There results show that not only atomic positions but also temperature factors can agree well in X-ray structures determined entirely independently by procedures which differ in virtually every possible technical aspect. A detailed comparison focussed on the effects of eglin binding on the structure of thermitase. Thermitase can be considered as consisting of (1) a central core of 94 residues, plus (2) four segments of 72 residues in total which shift as rigid bodies with respect to the core, plus (3) the remaining 113 residues which show small changes but, however, cannot be described as rigid bodies. The central cores of native thermitase and the 100 mM CaCl2 thermitase:eglin complex have an rms deviation of 0.13 A for 376 main chain atoms. One of the segments, formed by loops of the strong calcium binding site, shows differences up to 1.0 A in C alpha positions. These are probably due to crystal packing effects. The three other segments, comprising 51 residues, are affected conformational changes upon eglin binding so that the P1 to P3 binding pockets of thermitase broaden by 0.4 to 0.7 A. The residues involved in these changes correspond with residues which change position upon inhibitor binding in other subtilisins. This suggests that an induced fit mechanism is operational during substrate recognition by subtilisins.     

Interdomain compactization in human tyrosyl-tRNA synthetase studied by the hierarchical rotations technique

Aminoacyl-tRNA synthetases are key enzymes of protein biosynthesis which usually possess multidomain structures. Mammalian tyrosyl-tRNA synthetase is composed of two structural modules: N-terminal catalytic core and an EMAPII-like C-terminal domain separated by long flexible linker. The structure of full-length human cytoplasmic tyrosyl-tRNA synthetase is still unknown. The structures of isolated N-terminal and C-terminal domains of the protein are resolved, but their compact packing in a functional enzyme is a subject of debates. In this work we studied putative compactization of the N- and C-terminal modules of human tyrosyl-tRNA synthetase by the coarse-grained hierarchical rotations technique (HIEROT). The large number of distinct types of binding interfaces between N- and C-terminal modules is revealed in the absence of enzyme substrates. The binding propensities of different residues are computed and several binding "hot spots" are observed on the surfaces of N and C modules. These results could be used to govern atomistic molecular dynamics simulations, which will sample preferable binding interfaces effectively.     

Relating protein conformational changes to packing efficiency and disorder

Changes in protein conformation play key roles in facilitating various biochemical processes, ranging from signaling and phosphorylation to transport and catalysis. While various factors that drive these motions such as environmental changes and binding of small molecules are well understood, specific causative effects on the structural features of the protein due to these conformational changes have not been studied on a large scale. Here, we study protein conformational changes in relation to two key structural metrics: packing efficiency and disorder. Packing has been shown to be crucial for protein stability and function by many protein design and engineering studies. We study changes in packing efficiency during conformational changes, thus extending the analysis from a static context to a dynamic perspective and report some interesting observations. First, we study various proteins that adopt alternate conformations and find that tendencies to show motion and change in packing efficiency are correlated: residues that change their packing efficiency show larger motions. Second, our results suggest that residues that show higher changes in packing during motion are located on the changing interfaces which are formed during these conformational changes. These changing interfaces are slightly different from shear or static interfaces that have been analyzed in previous studies. Third, analysis of packing efficiency changes in the context of secondary structure shows that, as expected, residues buried in helices show the least change in packing efficiency, whereas those embedded in bends are most likely to change packing. Finally, by relating protein disorder to motions, we show that marginally disordered residues which are ordered enough to be crystallized but have sequence patterns indicative of disorder show higher dislocation and a higher change in packing than ordered ones and are located mostly on the changing interfaces. Overall, our results demonstrate that between the two conformations, the cores of the proteins remain mostly intact, whereas the interfaces display the most elasticity, both in terms of disorder and change in packing efficiency. By doing a variety of tests, we also show that our observations are robust to the solvation state of the proteins.     

The role of internal packing interactions in determining the structure and stability of a protein

Cassette mutagenesis has been used to investigate how internal packing interactions help to specify a protein's three-dimensional structure and stability. Three interacting residues in the hydrophobic core of the N-terminal domain of lambda repressor were randomized combinatorially. The randomization was restricted to the five amino acids Val, Leu, Ile, Met and Phe, thereby generating a sterically diverse set of core sequences composed solely of hydrophobic residues. We have isolated 78 of the 125 possible sequences generated by this randomization. Approximately 70% of the isolated sequences show some level of biological activity, and thus still carry sufficient information to encode the basic structure of lambda repressor. An assay based on the temperature dependence of activity in vivo has been used to estimate the relative activities and thermal stabilities of the set of mutants. In addition, nine mutants have been purified and their stabilities and DNA binding activities characterized in vitro. Of the 56 active sequences, only two, in addition to the wild-type, maintain the wild-type level of stability and activity. All three of these proteins satisfy stringent requirements for specifically shaped residues at each position. All of the remaining active sequences have reduced stabilities and/or reduced DNA binding affinities. These and previous results suggest that there are two levels of structural information encoded in core residues. At the first level, the basic structural information appears to reside largely in the hydrophobic character of these residues. The majority of sequences that simply maintain hydrophobicity at core positions are able to adopt the overall lambda repressor fold and maintain moderate stability. At the second, more detailed level, specific steric features of these residues and their packing interactions clearly act as important determinants of the protein's precise structure and stability. These results imply that many of the basic structural features of a protein could be predicted from relatively simple, degenerate sequence patterns.     

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.     

Core RNA polymerase from E. coli induces a major change in the domain arrangement of the sigma 70 subunit

Luminescence resonance energy transfer measurements were used to show that binding of E. coli core RNA polymerase induced major changes in interdomain distances in the sigma 70 subunit. The simplest model describing core-induced changes in sigma 70 involves a movement of the conserved region 1 by approximately 20 A and the conserved region 4.2 by approximately 15 A with respect to conserved region 2. The core-induced movement of region 1 (autoinhibition domain) and region 4.2 (DNA-binding domain) provides structural rationale for allosteric regulation of sigma 70 DNA binding properties by the core and suggests that this regulation may not only involve directly the autoinhibition domain of sigma 70 but also could involve a modulation of spacing between DNA-binding domains of sigma 70 induced by binding of core RNAP.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA.

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Generation of different nucleosome spacing periodicities in vitro. Possible origin of cell type specificity

We have been able to generate ordered nucleosome arrays that span the physiological range of spacing periodicities, using an in vitro system. Our system (a refinement of the procedure previously developed) uses the synthetic polynucleotide poly[d(A-T)], poly[d(A-T)], core histones, purified H1, and polyglutamic acid, a factor that increases nucleohistone solubility and greatly promotes the formation of ordered nucleosome arrays. This system has three useful features, not found in other chromatin assembly systems. First, it allowed us to examine histones from three different cell types/species (sea urchin sperm, chicken erythrocyte, and HeLa) as homologous or heterologous combinations of core and H1 histones. Second, it allowed us to control the average packing density (core histone to polynucleotide weight ratio) of nucleosomes on the polynucleotide; histone H1 is added in a second distinct step in the procedure to induce nucleosome alignment. Third, it permitted us to study nucleosome array formation in the absence of DNA base sequence effects. We show that the value of the spacing periodicity is controlled by the value of the initial average nucleosome packing density. The full range of physiological periodicities appears to be accessible to arrays generated using chicken erythrocyte (or HeLa) core histones in combination with chicken H5. However, chromatin-like structures cannot be assembled for some nucleosome packing densities in reactions involving some histone types, thus limiting the range of periodicities that can be achieved. For example, H1 histone types differ significantly in their ability to recruit disordered nucleosomes into ordered arrays at low packing densities. Sea urchin sperm H1 is more efficient than chicken H5, which is more efficient than H1 from HeLa or chicken erythrocyte. Sea urchin sperm core histones are more efficient in this respect than the other core histone types used. These findings suggest how different repeat lengths arise in different cell types and species, and provide new insights into the problems of nucleosome linker heterogeneity and how different types of chromatin structures could be generated in the same cell.     

Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val→Thr or Phe→Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Φ-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (> 2.5 kJ mol^− 1) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.     

THz time scale structural rearrangements and binding modes in lysozyme-ligand interactions

Predicting the conformational changes in proteins that are relevant for substrate binding is an ongoing challenge in the aim of elucidating the functional states of proteins. The motions that are induced by protein-ligand interactions are governed by the protein global modes. Our measurements indicate that the detected changes in the global backbone motion of the enzyme upon binding reflect a shift from the large-scale collective dominant mode in the unbound state towards a functional twisting deformation that assists in closing the binding cleft. Correlated motion in lysozyme has been implicated in enzyme function in previous studies, but detailed characterization of the internal fluctuations that enable the protein to explore the ensemble of conformations that ultimately foster large-scale conformational change is yet unknown. For this reason, we use THz spectroscopy to investigate the picosecond time scale binding modes and collective structural rearrangements that take place in hen egg white lysozyme (HEWL) when bound by the inhibitor (NAG) ₃. These protein thermal motions correspond to fluctuations that have a role in both selecting and sampling from the available protein intrinsic conformations that communicate function. Hence, investigation of these fast, collective modes may provide knowledge about the mechanism leading to the preferred binding process in HEWL-(NAG) ₃. Specifically, in this work we find that the picosecond time scale hydrogen-bonding rearrangements taking place in the protein hydration shell with binding modify the packing density within the hydrophobic core on a local level. These localized, intramolecular contact variations within the protein core appear to facilitate the large cooperative movements within the interfacial region separating the α- and β- domain that mediate binding. The THz time-scale fluctuations identified in the protein-ligand system may also reveal a molecular mechanism for substrate recognition.     

Methyl dynamics for understanding hydrophobic core packing of dynamically different motifs of double-stranded RNA binding domain of protein kinase R

Double-stranded RNA binding domains of human protein kinase R (dsRBD-PKR) regulate distinct cellular functions and the fate of an RNA molecule in the cell. This highly homologous domains present in multiple copies in a number of species, exhibit individual and specific functional specificity. Number of NMR and X-ray crystallographic structural studies reveals that such domains take a common alpha-beta-beta-beta-alpha tertiary fold. However, the functional specificities of these domains could be due to the dynamics of the individual amino acid residues, as has been shown earlier in the case of backbone dynamics of 15N-1H of dsRNA binding motifs (dsRBMs) of human protein kinase R (PKR) (Nanduri S, Rahman F, Williams BRG, Qin J. EMBO J 2000;19:5567-5574). To further investigate if the differences in dynamics of the two dsRBMs are restricted to only backbone, or if the side-chain motions are also different to the extent of influencing their packing of the two hydrophobic cores, we have investigated the methyl group dynamics using 13C-methyl relaxation measurements. The results show that the hydrophobic core of dsRBM1 is more tightly packed than dsRBM2, and it seems to undergo less fast scale motions in the subnanosecond regime.     

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.     

Pressure-induced conformational changes in a human Bence-Jones protein (Mcg)

The effect of high static pressures on the internal structure of the immunoglobulin light chain (Bence-Jones) dimer from the patient Mcg was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (less than 2 kbar), with a standard volume change of -93 mL/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis(8-anilino-naphthalene-1-sulfonate) (bis-ANS), rhodamine 123, and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the Mcg dimer exhibited a small decrease in affinity for bis-ANS (standard volume change approximately 5.9 mL/mol). At 3 kbar the binding activity increased by greater than 250-fold (volume change -144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures greater than 1.5 kbar (standard volume change -23 mL/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard volume change -5.5 mL/mol), but the protein was partially denatured at pressures greater than 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the Mcg dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Convergent and divergent ligand specificity among PDZ domains of the LAP and zonula occludens (ZO) families

We present a detailed comparative analysis of the PDZ domains of the human LAP proteins Erbin, Densin-180, and Scribble and the MAGUK ZO-1. Phage-displayed peptide libraries and in vitro affinity assays were used to define ligand binding profiles for each domain. The analysis reveals the importance of interactions with all four C-terminal residues of the ligand, which constitute a core recognition motif, and also the role of interactions with more upstream ligand residues that support and modulate the core binding interaction. In particular, the results highlight the importance of site(-1), which interacts with the penultimate residue of ligand C termini. Site(-1) was found to be monospecific in the Erbin PDZ domain (accepts tryptophan only), bispecific in the first PDZ domain of ZO-1 (accepts tryptophan or tyrosine), and promiscuous in the Scribble PDZ domains. Furthermore, it appears that the level of promiscuity within site(-1) greatly influences the range of potential biological partners and functions that can be associated with each protein. These findings show that subtle changes in binding specificity can significantly alter the range of biological partners for PDZ domains, and the insights enhance our understanding of this diverse family of peptide-binding modules.     

Alteration of the C-Terminal Ligand Specificity of the Erbin PDZ Domain by Allosteric Mutational Effects

Modulation of protein binding specificity is important for basic biology and for applied science. Here we explore how binding specificity is conveyed in PDZ (postsynaptic density protein-95/discs large/zonula occludens-1) domains, small interaction modules that recognize various proteins by binding to an extended C terminus. Our goal was to engineer variants of the Erbin PDZ domain with altered specificity for the most C-terminal position (position 0) where a Val is strongly preferred by the wild-type domain. We constructed a library of PDZ domains by randomizing residues in direct contact with position 0 and in a loop that is close to but does not contact position 0. We used phage display to select for PDZ variants that bind to 19 peptide ligands differing only at position 0. To verify that each obtained PDZ domain exhibited the correct binding specificity, we selected peptide ligands for each domain. Despite intensive efforts, we were only able to evolve Erbin PDZ domain variants with selectivity for the aliphatic C-terminal side chains Val, Ile and Leu. Interestingly, many PDZ domains with these three distinct specificities contained identical amino acids at positions that directly contact position 0 but differed in the loop that does not contact position 0. Computational modeling of the selected PDZ domains shows how slight conformational changes in the loop region propagate to the binding site and result in different binding specificities. Our results demonstrate that second-sphere residues could be crucial in determining protein binding specificity.