Structure, Function, and Thermodynamics of Lactate Dehydrogenases from Humans and the Malaria Parasite P. falciparum.

Temperature adaptation is ubiquitous among all living organisms, yet the molecular basis for this process remains poorly understood. It can be assumed that for parasite-host systems, the same enzymes found in both organisms respond to the same selection factor (human body temperature) with similar structural changes. Herein, we report the existence of a reversible temperature-dependent structural transition for the glycolytic enzyme lactate dehydrogenase (LDH) from the malaria parasite Plasmodium falciparum (pfLDH) and human heart (hhLDH) occurring in the temperature range of human fever. This transition is observed for LDHs from psychrophiles, mesophiles, and moderate thermophiles in their operating temperature range. Thermodynamic analysis reveals unique thermodynamic signatures of the LDH-substrate complexes defining a specific temperature range to which human LDH is adapted and parasite LDH is not, despite their common mesophilic nature. The results of spectroscopic analysis combined with the available crystallographic data reveal the existence of an active center within pfLDH that imparts psychrophilic structural properties to the enzyme. This center consists of two pockets, one formed by the five amino acids (5AA insert) within the substrate specificity loop and the other by the active site, that mutually regulate one another in response to temperature and induce structural and functional changes in the Michaelis complex. Our findings pave the way toward a new strategy for malaria treatments and drug design using therapeutic agents that inactivate malarial LDH selectively at a specific temperature range of the cyclic malaria paroxysm.

The Enthalpy-entropy Compensation Phenomenon. Limitations for the Use of Some Basic Thermodynamic Equations.

The thermodynamic analyses of proteins, protein-ligands and protein-nucleic acid complexes involves the entropy-enthalpy (S-H) compensation phenomenon. We have examined the question whether the observed compensation is artificial or reflects anything more than the well-known laws of statistical thermodynamics (so-called extra-thermodynamic compensation). We have shown that enthalpy- entropy compensation (EEC) is mainly the trivial consequence of the basic thermodynamic laws and there are no experimental evidences for existence of the extra-thermodynamic compensation. In most cases EEC obtained in the experiments through the plot enthalpies (ΔH) and entropies (TΔS) versus one another is meaningless due to the large correlated errors in ΔH and TΔS, unless special measures are taken to minimize, quantify and propagate these errors. Van't Hoff equation can be used for entropy calculation in limited cases when enthalpy is measured in independent experiments. Eyring equation cannot be used for calculation of entropy in any case and should be excluded from scientific use. Both equation, Van't Hoff and Eyring cannot be used for simultaneous calculation of the enthalpy and entropy values using one set of data. All the data obtained in this way should be recognized as erroneous.

Thermodynamic and Structural Adaptation Differences between the Mesophilic and Psychrophilic Lactate Dehydrogenases.

The thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (Sus scrofa; a mesophile), and mackerel icefish, cgLDH (Chamapsocephalus gunnari; a psychrophile), were investigated. Using a novel and quite sensitive fluorescence assay that can distinguish protein conformational changes close to and distal from the substrate binding pocket, a reversible global protein structural transition preceding the high-temperature transition (denaturation) was surprisingly found to coincide with a marked change in enzymatic activity for both LDHs. A similar reversible structural transition of the active site structure was observed for phLDH but not for cgLDH. An observed lower substrate binding affinity for cgLDH compared to that for phLDH was accompanied by a larger contribution of entropy to ΔG, which reflects a higher functional plasticity of the psychrophilic cgLDH compared to that of the mesophilic phLDH. The natural osmolyte, trimethylamine N-oxide (TMAO), increases stability and shifts all structural transitions to higher temperatures for both orthologs while simultaneously reducing catalytic activity. The presence of TMAO causes cgLDH to adopt catalytic parameters like those of phLDH in the absence of the osmolyte. Our results are most naturally understood within a model of enzyme dynamics whereby different conformations of the enzyme that have varied catalytic parameters (i.e., binding and catalytic proclivity) and whose population profiles are temperature-dependent and influenced by osmolytes interconvert among themselves. Our results also show that adaptation can be achieved by means other than gene mutations and complements the synchronic evolution of the cellular milieu.

MeCP2 Binding Cooperativity Inhibits DNA Modification-Specific Recognition.

Methyl-CpG binding protein 2 (MeCP2) is a multifunctional protein that guides neuronal development through its binding to DNA, recognition of sites of methyl-CpG (mCpG) DNA modification, and interaction with other regulatory proteins. Our study explores the relationship between mCpG and hydroxymethyl-CpG (hmCpG) recognition mediated by its mCpG binding domain (MBD) and binding cooperativity mediated by its C-terminal polypeptide. Previous study of the isolated MBD of MeCP2 documented an unusual mechanism by which ion uptake is required for discrimination of mCpG and hmCpG from CpG. MeCP2 binding cooperativity suppresses discrimination of modified DNA and is highly sensitive to both the total ion concentration and the type of counterions. Higher than physiological total ion concentrations completely suppress MeCP2 binding cooperativity, indicating a dominant electrostatic component to the interaction. Substitution of SO4(2-) for Cl(-) at physiological total ion concentrations also suppresses MeCP2 binding cooperativity, This effect is of particular note as the intracellular Cl(-) concentration changes during neuronal development. A related effect is that the protein-stabilizing solutes, TMAO and glutamate, reduce MeCP2 (but not isolated MBD) binding affinity by 2 orders of magnitude without affecting the apparent binding cooperativity. These observations suggest that polypeptide flexibility facilitates DNA binding by MeCP2. Consistent with this view, nuclear magnetic resonance (NMR) analyses show that ions have discrete effects on the structure of MeCP2, both MBD and the C-terminal domains. Notably, anion substitution results in changes in the NMR chemical shifts of residues, including some whose mutation causes the autism spectrum disorder Rett syndrome. Binding cooperativity makes MeCP2 an effective competitor with histone H1 for accessible DNA sites. The relationship between MeCP2 binding specificity and cooperativity is discussed in the context of chromatin binding, neuronal function, and neuronal development.

Unusual characteristics of the DNA binding domain of epigenetic regulatory protein MeCP2 determine its binding specificity.

The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity. The specificity for mCpG over CpG is >100-fold under these solution conditions. In contrast, the MBD does not discriminate hydroxymethyl-CpG from CpG. The MBD is unusual among site-specific DNA binding proteins in that (i) specificity is not conferred by the enhanced affinity for the specific site but rather by suppression of its affinity for generic DNA, (ii) its specific binding to mCpG is highly electrostatic, and (iii) it takes up as well as displaces monovalent cations upon DNA binding. The MBD displays an unusually high affinity for single-stranded DNA independent of modification or sequence. In addition, the MBD forms a discrete dimer on DNA via a noncooperative binding pathway. Because the affinity of the second monomer is 1 order of magnitude greater than that of nonspecific binding, the MBD dimer is a unique molecular complex. The significance of these results in the context of neuronal function and development and MeCP2-related developmental disorders such as Rett syndrome is discussed.

Stability, denaturation and refolding of Mycobacterium tuberculosis MfpA, a DNA mimicking protein that confers antibiotic resistance.

MfpA from Mycobacterium tuberculosis is a founding member of the pentapeptide repeat class of proteins (PRP) that is believed to confer bacterial resistance to the drug fluoroquinolone by mimicking the size, shape and surface charge of duplex DNA. We show that phenylalanine side chain stacking stabilizes the N-terminus of MfpA's pentapeptide thus extending the DNA mimicry analogy. The Lumry-Eyring model was applied to multiple spectral measures of MfpA denaturation revealing that the MfpA dimer dissociates to monomers which undergo a structural transition that leads to aggregation. MfpA retains high secondary and tertiary structure content under denaturing conditions. Dimerization stabilizes MfpA's pentapeptide repeat fold. The high Arrhenius activation energy of the barrier to aggregate formation rationalizes its stability. The mechanism of MfpA denaturation and refolding is a 'double funnel' energy landscape where the 'native' and 'aggregate' funnels are separated by the high barrier that is not overcome during in vitro refolding.

Circular dichroism spectroscopy has intrinsic limitations for protein secondary structure analysis.

Secondary structure content (SSC) cannot be calculated accurately from circular dichroism (CD) spectra for the majority of proteins whose three-dimensional structures have been solved. "Reliable" SSC that is significantly different from random SSC can be calculated from CD spectra only for all-alpha proteins and all-beta proteins with canonical beta-strand geometry.

Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA.

The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose beta-strands and turns within the right-handed quadrilateral beta-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.

DNA and protein footprinting analysis of the modulation of DNA binding by the N-terminal domain of the Saccharomyces cerevisiae TATA binding protein.

Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.

Influence of the N-terminal domain and divalent cations on self-association and DNA binding by the Saccharomyces cerevisiae TATA binding protein.

The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K+, and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the full-length protein.

Indirect readout of DNA sequence by papillomavirus E2 proteins depends upon net cation uptake.

The papillomavirus E2 proteins bind with high affinity to palindromic DNA sequences consisting of two highly conserved four base-pair sequences flanking a variable "spacer" of identical length (ACCG NNNN CGGT). While intimate contacts are observed between the bound proteins and conserved DNA in the available co-crystal structures, no contact is seen between the proteins and the spacer DNA. The ability of human papillomavirus strain 16 (HPV-16) E2 and bovine papillomavirus strain 1 (BPV-1) E2 to discriminate among binding sites with different spacer sequences is dependent on their sensitivity to the unique conformational and/or dynamic properties of the spacer DNA in a process termed "indirect readout". Differential sequence-specific K(+) uptake in low ionic strength solutions lacking Mg(2+) is observed upon E2 protein binding to sites containing the AATT, TTAA or ACGT spacer sequences. In contrast, the cation displacement typical of protein-DNA complex formation is observed at high K(+) concentrations or in the presence of Mg(2+). These results are interpreted to reflect the sequence-specific stabilization of bent DNA conformations by cations localized within the narrowed minor grooves of the protein-bound DNA and the intrinsic structure and flexibility of the DNA target. Mg(2+) differentially affects the binding of the HPV-16 E2 DNA binding domain (HPV16-E2/D) and the BPV-1 E2 DNA binding domain (BPV1-E2/D) to sites bearing different spacer sequences. This study suggests that monovalent and divalent cations contribute to the discrimination of DNA structure and flexibility that could in turn contribute to the specificity with which HPV16-E2/D and BPV1-E2/D mediate DNA replication and gene transcription.

Comparison of the effect of water release on the interaction of the Saccharomyces cerevisiae TATA binding protein (TBP) with "TATA Box" sequences composed of adenosine or inosine.

The formation of sequence-specific complexes of TATA binding protein (TBP) with the minor groove of DNA results in the burial of large nonpolar surfaces and the exclusion of water from these interfaces. The release of water is thus expected to provide a significant entropic driving force for formation of the transcription-preinitiated complexes mediated by the binding of TBP to specific sequences. In this article are described equilibrium-binding studies of Saccharomyces cerevisiae TBP to 14 bp oligonucleotides bearing either the tightly bound and efficiently transcribed adenovirus major late promoter (TATAAAAG) or its inosine-substituted derivative (TITIIIIG) as a function of neutral osmolyte concentration. These two DNA sequences present the same pattern of minor groove hydrogen-bond donors and acceptors to the protein. TBP-DNA complex formation was monitored by steady-state fluorescence resonance energy transfer measurements of the oligonucleotides end-labeled with fluorescein (donor) and TAMRA (acceptor). Correct interpretation of the results obtained with the inosine-substituted sequence required careful consideration of the optical properties of the dyes as a function of osmolyte concentration to demonstrate that the relative change in the end-to-end distances for TATAAAAG- and TITIIIIG-bearing oligonucleotides is the same upon TBP binding. Although the affinity of TBP is slightly greater for the adenosine compared with the inosine-substituted TATA sequence in the absence of osmolyte, the end-to-end distances of the bound DNA in complex with TBP, the enthalpic and electrostatic components of binding, are identical within experimental precision. However, approximately 18 additional molecules of water are released upon TBP binding the TATAAAAG as compared with the TITIIIIG sequence resulting in an entropic advantage to the binding of the natural promoter sequence. These results are considered with regard to differences in the flexibility and hydration of the two DNA sequences.

Solution structure and interdomain interactions of the Saccharomyces cerevisiae "TATA binding protein" (TBP) probed by radiolytic protein footprinting.

Although atomic-resolution crystal structures of the conserved C-terminal domain of several species of TBP and their complexes with DNA have been determined, little information is available concerning the structure in solution of full-length TBP containing both the conserved C-terminal and nonconserved N-terminal domains. Quantitation of the amino acid side chain oxidation products generated by synchrotron X-ray radiolysis by mass spectrometry has been used to determine the solvent accessibility of individual residues in monomeric Saccharomyces cerevisiae TATA binding protein (TBP) free in solution and in the TBP-DNA complex. Amino acid side chains within the C-terminal domain of unliganded full-length TBP that are predicted to be accessible from crystal structures of the isolated domain are protected from oxidation. Residues within the N-terminal domain are also protected from oxidation in both the absence and presence of DNA. Some residues within the DNA-binding "saddle" of the C-terminal domain are protected upon formation of a TBP-DNA complex as expected, while others are protected in both the absence and presence of bound DNA. In addition, residues on the upper side of the beta-sheets undergo reactivity changes as a function of DNA binding. These data suggest that the DNA-binding saddle of monomeric unliganded yeast TBP is only partially accessible to solvent, the N-terminal domain is partially structured, and the N- and C-terminal domains form a different set of contacts in the free and DNA-bound protein. The functional implications of these results are discussed.

Solution structural studies of the Saccharomyces cerevisiae TATA binding protein (TBP).

The intrinsic fluorescence of the six tyrosines located within the C-terminal domain of the Saccharomyces cerevisiae TATA binding protein (TBP) and the single tryptophan located in the N-terminal domain has been used to separately probe the structural changes associated with each domain upon DNA binding or oligomerization of the protein. The unusually short-wavelength maximum of TBP fluorescence is shown to reflect the unusually high quantum yield of the tyrosine residues in TBP and not to result from unusual tryptophan fluorescence. The anisotropy of the C-terminal tyrosines is very high in monomeric, octameric, and DNA-complexed TBP and comparable to that observed in much larger proteins. The tyrosines have low accessibility to an external fluorescence quencher. The anisotropy of the single tryptophan located within the N-terminal domain of TBP is much lower than that of the tyrosines and is accessible to an external fluorescence quencher. Tyrosine, but not tryptophan, fluorescence is quenched upon TBP-DNA complex formation. Only the tryptophan fluorescence is shifted to longer wavelengths in the protein-DNA complex. In addition, the accessibility of the tryptophan residue to the external quencher and the internal motion of the tryptophan residue increase upon DNA binding by TBP. These results show the following: (i) The structure of the C-terminal domain structure is unchanged upon TBP oligomerization, in contrast to the N-terminal domain [Daugherty, M. A., Brenowitz, M., and Fried, M. G. (2000) Biochemistry 39, 4869-4880]. (ii) The environment of the tyrosine residues within the C-terminal domain of TBP is structurally rigid and unaffected by oligomerization or DNA binding. (iii) The C-terminal domain of TBP is uniformly in close proximity to bound DNA. (iv) While the N-terminal domain unfolds upon DNA binding by TBP, its increased correlation time shows that the overall structure of the protein is more rigid when complexed to DNA. A model that reconciles these results is proposed.