Integrating Neuropsychology and Brain Imaging for a Referral of Possible Pseudodementia: A Case Report.

The study aimed to highlight the importance of interdisciplinary collaboration and the value for combining normative neuropsychological and neuroradiological measures for clinical purposes. We present the case of "CL," a 65-year-old, right-handed, Caucasian female referred for a neuropsychological evaluation of memory difficulties and depression with the rule-out of pseudodementia. A brain magnetic resonance imaging (MRI) scan was conducted within 24 hours of the neuropsychology exam. Mood measures showed elevated depression and apathy symptoms. The neuropsychological profile showed variable effort, intact comprehension but compromised confrontation naming and verbal memory deficits. Using normative references from 20 female age- and education-matched healthy control peers, CL showed significantly reduced temporal cortex thickness with reduced bilateral hippocampal, right amygdala, and right caudate volumes. Combined data were supportive of a diagnosis of semantic dementia. Examining neuropsychological profiles in combination with neuroimaging standardized metrics relative to peers improved case conceptualization. Standard measures of effort and malingering examined alone and without MRI for the diagnosis of pseudodementia have questionable validity and rationale. We additionally discuss the advantages and limitations/challenges for integrating neuropsychological assessments with normative based MRI brain metrics.

Desulfovibrio desulfuricans G20 tetraheme cytochrome structure at 1.5 Angstrom and cytochrome interaction with metal complexes.

The structure of the type I tetraheme cytochrome c(3) from Desulfovibrio desulfuricans G20 was determined to 1.5 Angstrom by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated by circular dichroism studies. Again, only small changes in protein structure were documented. The location of the molybdate ion near heme IV in the crystal structure suggested heme IV as the site of electron exit from the reduced cytochrome and implicated Lys14 and Lys56 in binding. Analysis of structurally conserved water molecules in type I cytochrome c(3) crystal structures identified interactions predicted to be important for protein stability and possibly for intramolecular electron transfer among heme molecules.

Crystal structure of an antigen-binding fragment bound to single-stranded DNA.

Antibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus (SLE) and they also serve as models for the study of protein-DNA recognition. Anti-DNA antibodies often play an important role in disease pathogenesis by mediating kidney damage via antibody-DNA immune complex formation. The structural underpinnings of anti-DNA antibody pathogenicity and antibody-DNA recognition, however, are not well understood, due in part to the lack of direct, experimental three-dimensional structural information on antibody-DNA complexes. To address these issues for anti-single-stranded DNA antibodies, we have determined the 2.1 A crystal structure of a recombinant Fab (DNA-1) in complex with dT5. DNA-1 was previously isolated from a bacteriophage Fab display library from the immunoglobulin repertoire of an SLE-prone mouse. The structure shows that DNA-1 binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds. The critical stacking Tyr residues are L32, L49, H100, and H100A, while His L91 and Asn L50 contribute hydrogen bonds. Comparison of the DNA-1 structure to other anti-nucleic acid Fab structures reveals a common ssDNA recognition module consisting of Tyr L32, a hydrogen bonding residue at position L91, and an aromatic side-chain from the tip of complementarity determining region H3. The structure also provides a framework for interpreting previously determined thermodynamics data, and this analysis suggests that hydrophobic desolvation might underlie the observed negative enthalpy of binding. Finally, Arg side-chains from complementarity determining region H3 appear to play a novel role in DNA-1. Rather than forming ion pairs with dT5, Arg contributes to oligo(dT) recognition by helping to maintain the structural integrity of the combining site. This result is significant because antibody pathogenicity is thought to be correlated to the Arg content of anti-DNA antibody hypervariable loops.

Crystallization and preliminary crystallographic analysis of the proline dehydrogenase domain of the multifunctional PutA flavoprotein from Escherichia coli.

The PutA flavoprotein from Escherichia coli is a multifunctional protein that plays pivotal roles in proline catabolism by functioning as both a membrane-associated bifunctional enzyme and a transcriptional repressor. Peripherally membrane-bound PutA catalyzes the two-step oxidation of proline to glutamate, while cytoplasmic PutA represses the transcription of its own gene and the gene for a proline-transporter protein. X-ray crystallographic studies on PutA have been initiated to determine how the PutA structural scaffold enables it to be both an enzyme and a repressor, and to understand the mechanism by which PutA switches between its enzymatic and DNA-binding functions. To facilitate crystallization, a recombinant protein (PutA669) corresponding to the N-terminal 669 amino-acid residues of the 1320 residues of PutA was engineered. Activity assays demonstrated that PutA669 catalyzes the first step of chemistry performed by PutA, the conversion of proline to Delta1-pyrroline-5-carboxylate. Crystals of PutA669 have been obtained from PEG 3000 buffered at pH 6-7. The crystals occupy an I-centered orthorhombic lattice with unit-cell parameters a = 72.5, b = 140.2, c = 146.8 A; a 2.15 A data set was collected using a rotating-anode source. Assuming one molecule per asymmetric unit, the Matthews coefficient V(M) is 2.5 A(3) Da(-1), with a solvent content of 50%. The structure of PutA669 will be solved by multiple isomorphous replacement.

Crystallization and molecular-replacement studies of a recombinant antigen-binding fragment complexed with single-stranded DNA.

Anti-DNA antibodies have been implicated in autoimmune diseases and also serve as models for understanding protein-DNA recognition. Crystals of a recombinant antigen-binding fragment (Fab) complexed with dT(5) have been obtained and initial phases have been determined using molecular replacement. The crystals diffract to 2.1 A resolution and occupy space group P6(5)22, with unit-cell parameters a = 171.8, c = 144.6 A; there are two Fabs per asymmetric unit. X-PLORdirect rotation-function calculations followed by Patterson correlation filtering were successful when using a Fab search model; however, they failed when using the individual variable and conserved domains of the Fab as search models. AMoRe successfully identified the correct solution in cases where X-PLOR failed.

Conformations of nicotinamide adenine dinucleotide (NAD(+)) in various environments.

Enzymes bind NAD(+) in extended conformations and yet NAD(+) exists in aqueous solution as a compact, folded molecule. Thus, NAD(+) conformation is environment dependent. In an attempt to investigate the effects of environmental changes on the conformation of NAD(+), a series of molecular dynamics simulations in different solvents was performed. The solvents investigated (water, DMSO, methanol and chloroform) represented changes in relative permittivity and hydrophobic character. The simulations predicted folded conformations of NAD(+) to be more stable in water, DMSO and methanol. In contrast, extended conformations of NAD(+) were observed to be more stable in chloroform. Furthermore, the extended conformations observed in chloroform were similar to conformations of NAD(+) bound to enzymes. In particular, a large separation between the aromatic rings and a strong interaction between the pyrophosphate and nicotinamide groups were observed. The implications of these observations for the recognition of NAD(+) by enzymes is discussed. It is argued that a hydrophobic environment is important for stabilizing unfolded conformations of NAD(+).

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).

Structure of bacterial luciferase beta 2 homodimer: implications for flavin binding.

The crystal structure of the beta 2 homodimer of Vibrio harveyi luciferase has been determined to 2.5 A resolution by molecular replacement. Crystals were grown serendipitously using the alpha beta form of the enzyme. The subunits of the homodimer share considerable structural homology to the beta subunit of the alpha beta luciferase heterodimer. The four C-terminal residues that are disordered in the alpha beta structure are fully resolved in our structure. Four peptide bonds have been flipped relative to their orientations in the beta subunit of the alpha beta structure. The dimer interface of the homodimer is smaller than the interface of the heterodimer in terms of buried surface area and number of hydrogen bonds and salt links. Inspection of the subunits of our structure suggests that FMNH2 cannot bind to the beta 2 enzyme at the site that has been proposed for the alpha beta enzyme. However, we do uncover a potential FMNH2 binding pocket in the dimer interface, and we model FMN into this site. This proposed flavin binding motif is consistent with several lines of biochemical and structural evidence and leads to several conclusions. First, only one FMNH2 binds per homodimer. Second, we predict that reduced FAD and riboflavin should be poor substrates for beta 2. Third, the reduced activity of beta 2 compared to alpha beta is due to solvent exposure of the isoalloxazine ring in the beta 2 active site. Finally, we raise the question of whether our proposed flavin binding site could also be the binding site for flavin in the alpha beta enzyme.

Flavin reductase P: structure of a dimeric enzyme that reduces flavin.

We report the structure of an NADPH:FMN oxidoreductase (flavin reductase P) that is involved in bioluminescence by providing reduced FMN to luciferase. The 1.8 A crystal structure of flavin reductase P from Vibrio harveyi was solved by multiple isomorphous replacement and reveals that the enzyme is a unique dimer of interlocking subunits, with 9352 A2 of surface area buried in the dimer interface. Each subunit comprises two domains. The first domain consists of a four-stranded antiparallel beta-sheet flanked by helices on either side. The second domain reaches out from one subunit and embraces the other subunit and is responsible for interlocking the two subunits. Our structure explains why flavin reductase P is specific for FMN as cofactor. FMN is recognized and tightly bound by a network of 16 hydrogen bonds, while steric considerations prevent the binding of FAD. A flexible loop containing a Lys and an Arg could account for the NADPH specificity. The structure reveals information about several aspects of the catalytic mechanism. For example, we show that the first step in catalysis, which is hydride transfer from C4 of NADPH to cofactor FMN, involves addition to the re face of the FMN, probably at the N5 position. The limited accessibility of the FMN binding pocket and the extensive FMN-protein hydrogen bond network are consistent with the observed ping-pong bisubstrate--biproduct reaction kinetics. Finally, we propose a model for how flavin reductase P might shuttle electrons between NADPH and luciferase.

Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 25 Angstroms Resolution.

The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution. To study the determinants of thermostability, we compare our structure to four other GAPDHs. Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed. We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners. These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links. The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds. Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner. Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles. There is, however, an association between thermostability and decreasing enzyme surface to volume ratio. Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs. These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues.

Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: evidence for an uncoupled R state.

In the R form of ATCase complexed with the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, large temperature factors are reported for the allosteric domains of the regulatory chains. We studied the conformational flexibility of the holoenzyme with molecular dynamics simulations and rigid body (TLS) analysis. The results of the molecular dynamics simulations suggest that, although local atomic fluctuations account for the temperature factors of the catalytic and zinc domains, they do not account for the large temperature factors of the allosteric regions. However, the temperature factors of the allosteric domains can be satisfactorily analyzed using a rigid body model. The simulations and rigid body analysis support the idea that the allosteric regions are mechanically uncoupled from the rest of the enzyme in the PALA structure. Implications of this uncoupling for allosteric regulation are discussed.

Anti-insulin antibody structure and conformation. II. Molecular dynamics with explicit solvent.

Molecular dynamics at 300 K was used as a conformation searching tool to analyze a knowledge-based structure prediction of an anti-insulin antibody. Solvation effects were modeled by packing water molecules around the antigen binding loops. Some loops underwent backbone and side-chain conformational changes during the 95-ps equilibration, and most of these new, lower potential energy conformations were stable during the subsequent 200-ps simulation. Alterations to the model include changes in the intraloop, main-chain hydrogen bonding network of loop H3, and adjustments of Tyr and Lys side chains of H3 induced by hydrogen bonding to water molecules. The structures observed during molecular dynamics support the conclusion of the previous paper that hydrogen bonding will play the dominant role in antibody-insulin recognition. Determination of the structure of the antibody by x-ray crystallography is currently being pursued to provide an experimental test of these results. The simulation appears to improve the model, but longer simulations at higher temperatures should be performed.