bluuebunny/crossref_metadata_2025 huggingface dataset:
Free energy determinants of tertiary structure and the evaluation of protein models
Authors: Petrey Donald , Honig Barry
Published: February 2009

We develop a protocol for estimating the free energy difference between different conformations of the same polypeptide chain. The conformational free energy evaluation combines the CHARMM force field with a continuum treatment of the solvent. In almost all cases studied, experimentally determined structures are predicted to be more stable than misfolded “decoys.” This is due in part to the fact that the Coulomb energy of the native protein is consistently lower than that of the decoys. The solvation free energy generally favors the decoys, although the total electrostatic free energy (sum of Coulomb and solvation terms) favors the native structure. The behavior of the solvation free energy is somewhat counterintuitive and, surprisingly, is not correlated with differences in the burial of polar area between native structures and decoys. Rather, the effect is due to a more favorable charge distribution in the native protein, which, as is discussed, will tend to decrease its interaction with the solvent. Our results thus suggest, in keeping with a number of recent studies, that electrostatic interactions may play an important role in determining the native topology of a folded protein. On this basis, a simplified scoring function is derived that combines a Coulomb term with a hydrophobic contact term. This function performs as well as the more complete free energy evaluation in distinguishing the native structure from misfolded decoys. Its computational efficiency suggests that it can be used in protein structure prediction applications, and that it provides a physically well‐defined alternative to statistically derived scoring functions.
Three‐dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: New insights into the activation process
Authors: Bessis Anne‐Sophie , Acher Francine , Bertrand Hugues‐Olivier , Galvez Thierry , De Colle Cyril , Pin Jean‐Philippe
Published: February 2009

Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G‐protein‐coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7‐transmembrane domains required for G‐protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R). The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine‐binding protein (LIVBP) and leucine‐binding protein (LBP) as already known, and acetamide‐binding protein (AmiC). These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft. Models for both open and closed forms of mGlu4R ATD have been generated. ACPT‐I (1‐aminocyclopentane 1,3,4‐tricarboxylic acid), a selective agonist, has been docked in the two models. In the open form, ACPT‐I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182. In the closed form, ACPT‐I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II. These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.
Enhanced internal dynamics of a membrane transport protein during substrate translocation
Authors: Döring KLAUS , Surrey Thomas , Grünewald Sylvia , John Edgar , Jähnig Fritz
Published: February 2009

Conformational changes are essential for the activity of many proteins. If, or how fast, internal fluctuations are related to slow conformational changes that mediate protein function is not understood. In this study, we measure internal fluctuations of the transport protein lactose permease in the presence and absence of substrate by tryptophan fluorescence spectroscopy. We demonstrate that nanosecond fluctuations of α‐helices are enhanced when the enzyme transports substrate. This correlates with previously published kinetic data from transport measurements showing that millisecond conformational transitions of the substrate‐loaded carrier are faster than those in the absence of substrate. These findings corroborate the hypothesis of the hierarchical model of protein dynamics that predicts that slow conformational transitions are based on fast, thermally activated internal motions.
Exploring steric constraints on protein mutations using MAGE/PROBE
Authors: Word J. Michael , Presley Brent K. , Lovell Simon C. , Richardson David C. , Batemanm Robert C.
Published: February 2009

When planning a mutation to test some hypothesis, one crucial question is whether the new side chain is compatible with the existing structure; only if it is compatible can the interpretation of mutational results be straightforward. This paper presents a simple way of using the sensitive geometry of all‐atom contacts (including hydrogens) to answer that question. The interactive MAGE/PROBE system lets the biologist explore conformational space for the mutant side chain, with an interactively updated kinemage display of its all‐atom contacts to the original structure. The Autobondrot function in PROBE systematically explores that same conformational space, outputting contact scores at each point, which are then contoured and displayed. These procedures are applied here in two types of test cases, with known mutant structures. In ricin A chain, the ability of a neighboring glutamate to rescue activity of an active‐site mutant is modeled successfully. In T4 lysozyme, six mutations to Leu are analyzed within the wild‐type background structure, and their Autobondrot score maps correctly predict whether or not their surroundings must shift significantly in the actual mutant structures; interactive examination of contacts for the conformations involved explains which clashes are relieved by the motions. These programs are easy to use, are available free for UNIX or Microsoft Windows operating systems, and should be of significant help in choosing good mutation experiments or in understanding puzzling results.
Evaluation of PSI‐BLAST alignment accuracy in comparison to structural alignments
Authors: Friedberg Iddo , Kaplan Tommy , Margalit Hanah
Published: February 2009

The PSI‐BLAST algorithm has been acknowledged as one of the most powerful tools for detecting remote evolutionary relationships by sequence considerations only. This has been demonstrated by its ability to recognize remote structural homologues and by the greatest coverage it enables in annotation of a complete genome. Although recognizing the correct fold of a sequence is of major importance, the accuracy of the alignment is crucial for the success of modeling one sequence by the structure of its remote homologue. Here we assess the accuracy of PSI‐BLAST alignments on a stringent database of 123 structurally similar, sequence‐dissimilar pairs of proteins, by comparing them to the alignments defined on a structural basis. Each protein sequence is compared to a nonredundant database of the protein sequences by PSI‐BLAST. Whenever a pair member detects its pair‐mate, the positions that are aligned both in the sequential and structural alignments are determined, and the alignment sensitivity is expressed as the per‐centage of these positions out of the structural alignment. Fifty‐two sequences detected their pair‐mates (for 16 pairs the success was bi‐directional when either pair member was used as a query). The average percentage of correctly aligned residues per structural alignment was 43.5 ± 2.2%. Other properties of the alignments were also examined, such as the sensitivity vs. specificity and the change in these parameters over consecutive iterations. Notably, there is an improvement in alignment sensitivity over consecutive iterations, reaching an average of 50.9 + 2.5% within the five iterations tested in the current study.
Prediction of a common β‐propeller catalytic domain for fructosyltransferases of different origin and substrate specificity
Authors: Pons Tirso , Hernández Lázaro , Batista Frank R. , Chinea Glay
Published: February 2009

The three‐dimensional (3D) structure of fructan biosyn‐thetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence‐structure compatibility search using TOPITS, SDP, 3D‐PSSM, and SAM‐T98 programs identified a β‐propeller fold with scores above the confidence thresh‐old that indicate a structurally conserved catalytic domain in fruc‐tosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence–sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern {H‐x(2)‐[PTV]‐x(4)‐[LIVMA]‐[NSCAYG]‐[DE]‐P‐[NDSC]‐[GA]} for this family.
Design of a minimal protein oligomerization domain by a structural approach
Authors: Burkhard Peter , Meier M. , Lustig Ariel
Published: February 2009

Because of the simplicity and regularity of the α‐helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad‐repeat peptide stabilized by a complex network of inter‐ and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly α‐helical and 100% dimeric under physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X‐ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native‐like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.
Cysteine‐independent polymerization of metallothioneins in solutions and in crystals
Authors: Hou Tingjun , Xu Xiaojie , An Yu , Ru Binggen , Bi Ruchang
Published: February 2009

Polymerization of metallothioneins is one of the usually encountered puzzles during the research process of metallothioneins' structure and function. Our work focuses on the cysteine independently occurred polymerization from metallothioneins monomers in different milieus, while it leaves out the aggregation caused by the oxidation of cysteine, because the latter circumstance is the result of purification lapsus. After the purification of metallothioneins monomers, a dynamic light‐scattering technique is used to detect the polymerized states of rabbit liver metallothionein I and II in different buffers, which is the first systematical detection of polymerized states of metallothioneins in solutions. The effects of different compositions of each buffer are discussed in details. Steric complementarity, hydrophobic, and electrostatic interaction characteristics are studied, following the modeling of monomers and relevant polymers of rat metallothionein II, rabbit liver metallothionein I and II. These theoretical calculations are the first complete computer simulations on different factors affecting metallothioneins' polymerization. A molecular recognition mechanism of metallothioneins' polymerization in solutions is proposed on the bases of experimental results and theoretical calculations. Preliminary X‐ray studies of two crystal forms of rabbit liver metallothionein II are compared with the crystal structure of rat metallothionein II, and the polymerized states in crystal packing are discussed with the knowledge of polymerization of metallothioneins in solutions. The hypothesis, which is consistent with theoretical calculations and experimental results, is expected to construct a connection between the biochemical characteristics and physiological functions of metallothioneins, and this research may give some enlightenment to the topics of protein polymerizations.
β‐Helix core packing within the triple‐stranded oligomerization domain of the P22 tailspike
Authors: Kreisberg Jason F. , Betts Scott D. , King Jonathan
Published: February 2009

A right‐handed parallel β‐helix of 400 residues in 13 tightly packed coils is a major motif of the chains forming the trimeric P22 tailspike adhesin. The β‐helix domains of three identical subunits are side‐by‐side in the trimer and make predominantly hydrophilic inter‐subunit contacts (Steinbacher S et al., 1994, Science 265:383‐386). After the 13th coil the three individual β‐helices terminate and the chains wrap around each other to form three interdigitated β‐sheets organized into the walls of a triangular prism. The β‐strands then separate and form antiparallel β‐sheets, but still defining a triangular prism in which each side is a β‐sheet from a different subunit (Seckler R, 1998, J Struct Biol 122:216–222). The subunit interfaces are buried in the triangular core of the prism, which is densely packed with hydrophobic side chains from the three β‐sheets. Examination of this structure reveals that its packed core maintains the same pattern of interior packing found in the left‐handed β‐helix, a single‐chain structure. This packing is maintained in both the interdigitated parallel region of the prism and the following antiparallel sheet section. This oligomerization motif for the tailspike β‐helices presumably contributes to the very high thermal and detergent stability that is a property of the native tailspike adhesin.
Stability and peptide binding specificity of Btk SH2 domain: Molecular basis for X‐linked agammaglobulinemia
Authors: Tzeng Shiou‐Ru , Pai Ming‐Tao , Wu Chih‐Wei , Cheng Jya‐Wei , Lung Feng‐Di T. , Roller Peter P. , Lei Benfang , Wei Chih‐Jen , Tu Shiao‐Chun , Chen Shi‐Han , Soong Wen‐Jue
Published: February 2009

X‐linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B‐cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C‐γ phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B‐cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C‐γ. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B‐cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and I370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200‐fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and I370 mutations in the pY + 3 hydrophobic binding pocket (∼3‐ to 17‐folds). Furthermore, mutation of the Tyr residue at the βD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.
Resonant mirror biosensor analysis of type Iα cAMP‐dependent protein kinase B domain—Cyclic nucleotide interactions
Authors: Muhonen Wallace W. , Shabb John B.
Published: February 2009

A resonant mirror biosensor was used to study cyclic nucleotide‐receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide‐binding properties of the wild‐type isolated B domain of the cAMP‐dependent protein kinase type Iα regulatory subunit and its Ala‐334‐Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP‐saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X‐100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild‐type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor‐determined kinetic constants. Biosensor‐determined relative inhibition constants (K'i = KicAMP/K'ianalog) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published K'i values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The K'i values for the wild‐type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide‐receptor interactions.
The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis
Authors: Rath Arianna , Davidson Alan R.
Published: February 2009

We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 °C and a ΔGu of 3.08 kcal/mol. Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild‐type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment. Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 °C with concomitant increases in ΔGu of up to 1.4 kcal/mol. The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 °C and a ΔGu more than double that of the wild‐type domain. The folding rate of this hyperstable mutant was 10‐fold faster than wild‐type, while its unfolding rate was fivefold slower. All of the stabilized mutants were still able to bind a target peptide with wild‐type affinity. We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies. In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.
Thirty‐plus functional families from a single motif
Authors: Yu Lihua , Smith Temple F. , Gaitatzes Chrysanthe
Published: February 2009

It is now possible to identify over 30 functional subfamilies among the WD‐repeat‐containing proteins found in the completed genomes. The majority of these subfamilies have at least one member for which experimental data allow assignment to a cellular pathway or process. Half of the 63 WD‐repeat‐containing proteins in Saccharomyces cerevisiae, half of the 70 in Caenorhabditis elegans, and a third of the 100 plus predicted in Drosophila can be assigned to 23 of these functional subfamilies. Perhaps indicative of the future, 33 WD‐repeat‐containing proteins from the partial genome of Arabidopsis thaliana can now be assigned to 18 of these subfamilies. These assignments have been made possible by combining traditional sequence similarity with an implied common beta propeller structural context to obtain measures of protein‐protein surface similarity. The beta propeller structural context is represented in the form of a Hidden Markov Model. The procedure is completely automated.
Structural changes in α‐synuclein affect its chaperone‐like activity in vitro
Authors: Kim Thomas D. , Paik Seung R. , Yang Chul‐Hak , Kim Jongsun
Published: February 2009

α‐Synuclein, a major constituent of Lewy bodies (LBs) in Parkinson's disease (PD), has been implicated to play a critical role in synaptic events, such as neuronal plasticity during development, learning, and degeneration under pathological conditions, although the physiological function of α‐synuclein has not yet been established. We here present biochemical evidence that recombinant α‐synuclein has a chaperone‐like function against thermal and chemical stress in vitro. In our experiments, α‐synuclein protected glutathione S‐transferase (GST) and aldolase from heat‐induced precipitation, and α‐lactalbumin and bovine serum albumin from dithiothreitol (DTT)‐induced precipitation like other molecular chaperones. Moreover, preheating of α‐synuclein, which is believed to reorganize the molecular surface of α‐synuclein, increased the chaperone‐like activity. Interestingly, in organic solvents, which promotes the formation of secondary structure, α‐synuclein aggregated more easily than in its native condition, which eventually might abrogate the chaperone‐like function of the protein. In addition, α‐synuclien was also rapidly and significantly precipitated by heat in the presence of Zn2+in vitro, whereas it was not affected by the presence of Ca2+or Mg2+. Circular dichroism spectra confirmed that α‐synuclein underwent conformational change in the presence of Zn2+. Taken together, our data suggest that α‐synuclein could act as a molecular chaperone, and that the conformational change of the α‐synuclein could explain the aggregation kinetics of α‐synuclein, which may be related to the abolishment of the chaperonic‐like activity.
Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: Overview and feasibility: Properties of diazirine as a precursor
Authors: Richards Frederic M. , Lamed Raphael , Wynn Richard , Patel Darshan , Olack Gerard
Published: February 2009

Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C‐H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra : CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble.
An improved tripod amphiphile for membrane protein solubilization
Authors: Yu Seungju M. , McQuade D. Tyler , Quinn Mariah A. , Hackenberger Christian P.R. , Gellman Samuel H. , Krebs Mark P. , Polans Arthur S.
Published: February 2009

Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a “tripod amphiphile,” that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine‐oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native‐like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long‐range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.
Ab initio molecular dynamics studies on HIV‐1 reverse transcriptase triphosphate binding site: Implications for nucleoside‐analog drug resistance
Authors: Alber Frank , Carloni Paolo
Published: February 2009

Quantum‐chemical methods are used to shed light on the functional role of residues involved in the resistance of HIV‐1 reverse transcriptase against nucleoside‐analog drugs. Ab initio molecular dynamics simulations are carried out for models representing the adduct between the triphosphate substrate and the nucleoside binding site. The triphosphate is considered either deprotonated or protonated at the γ‐position. Although the protonated form already experiences large rearrangements in the ps time scale, the fully deprotonated state exhibits a previously unrecognized low‐barrier hydrogen bond between Lys65 and γ‐phosphate. Absence of this interaction in Lys65→Arg HIV‐1 RT might play a prominent role in the resistance of this mutant for nucleoside analogs (Gu Z et al., 1994b, Antimicrob Agents Chemother 38:275–281; Zhang D et al., 1994, Antimicrob Agents Chemother 38:282–287). Water molecules present in the active site, not detected in the X‐ray structure, form a complex H‐bond network. Among these waters, one may be crucial for substrate recognition as it bridges Gln151 and Arg72 with the β‐phosphate. Absence of this stabilizing interaction in Gln151→Met HIV‐1 RT mutant may be a key factor for the known drug resistance of this mutant toward dideoxy‐type drugs and AZT (Shirasaka T et al., 1995, Proc Natl Acad Sci USA 92:2398–2402; Iversen AK et al., 1996, J Virol 70:1086–1090).
N‐terminal mutations in the anti‐estradiol Fab 57‐2 modify its hapten binding properties
Authors: Saviranta Petri , Jauria Piitu , Lamminmäki Urpo , Eriksson Susann , Lövgren Timo , Hellman Jukka
Published: February 2009

Recombinant antibodies often contain N‐terminal mutations arising from the use of degenerate cloning primer sets and/or the introduction of restriction sites in the framework 1 regions. We studied the effects of such mutations in a recombinant anti‐estradiol Fab fragment derived from the hybridoma cell line 57‐2. The 5′ ends of the heavy and light chain genes were originally modified to introduce the restriction sites XhoI and SacI, respectively, for cloning purposes. However, the affinity and specificity of the recombinant Fab were lowered compared to the proteolytic Fab' fragment of the parental hybridoma IgG. Replacing the mutated sites with authentic amino acid coding sequences restored the binding properties as well as increased the bacterial production levels fivefold and 10‐fold at 30 and 37°C, respectively. Local changes in the antigen binding site were probed by determining the affinity constants (Ka) for estradiol and four related steroids. It was found that the mutated heavy chain amino terminus specifically increased the Ka for testosterone whereas the mutated light chain amino terminus decreased the Ka for all of the steroids to the same extent; the heavy and light chain effects were additive. Analysis of a newly determined crystal structure of the authentic Fab 57‐2 in complex with estradiol suggests that mutations in the residue 2 in VH, and 2 and 4 in the VL domain were those responsible for the observed effects. Their general roles as structure‐determining residues for the CDR3 loops imply that similar effects can occur with other recombinant antibodies as well.
For the record: A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica
Authors: Smooker Peter M. , Whisstock James C. , Irving James A. , Siyaguna Sugeeshwara , Spithill Terry W. , Pike Robert N.
Published: February 2009

The trematode Fasciola hepatica secretes a number of cathepsin L‐like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.
For the record: Fluorescence and 19F NMR evidence that phenylalanine, 3‐L‐fluorophenylalanine and 4‐L‐fluorophenylalanine bind to the L‐leucine specific receptor of Escherichia coli
Authors: Luck Linda A. , Johnson Colin
Published: February 2009

The binding capacity of the L‐leucine receptor from Escherichia coli was measured with L‐phenylalanine and 4‐fluoro‐L‐phenylalanine as substrates by fluorescence. The apparent dissociation constants (KD) for L‐leucine, L‐phenylalanine, and 4‐fluoro‐L‐phenylalanine are 0.40, 0.18, and 0.26 respectively. 19F NMR data show protein‐induced shifts for the 4‐fluoro‐L‐phenylalanine peak and 3‐fluoro‐L‐phenylalanine when receptor is present. Evidence points to the binding of only the L‐isomers of these fluorine analogs.
Post‐translational modification is essential for catalytic activity of nitrile hydratase
Authors: Murakami Taku , Nojiri Masaki , Nakayama Hiroshi , Dohmae Naoshi , Takio Koji , Odaka Masafumi , Endo Isao , Nagamune Teruyuki , Yohda Masafumi
Published: February 2009

Nitrile hydratase from Rhodococcus sp. N‐771 is an αβ heterodimer with a nonheme ferric iron in the catalytic center. In the catalytic center, αCys112 and αCys114 are modified to a cysteine sulfinic acid (Cys‐SO2H) and a cysteine sulfenic acid (Cys‐SOH), respectively. To understand the function and the biogenic mechanism of these modified residues, we reconstituted the nitrile hydratase from recombinant unmodified subunits. The αβ complex reconstituted under argon exhibited no activity. However, it gradually gained the enzymatic activity through aerobic incubation. ESI‐LC/MS analysis showed that the anaerobically reconstituted αβ complex did not have the modification of αCys112‐SO2H and aerobic incubation induced the modification. The activity of the reconstituted αβ complex correlated with the amount of αCys112‐SO2H. Furthermore, ESI‐LC/MS analyses of the tryptic digest of the reconstituted complex, removed of ferric iron at low pH and carboxamidomethylated without reduction, suggested that αCys114 is modified to Cys‐SOH together with the sulfinic acid modification of αCys112. These results suggest that αCys112 and αCys114 are spontaneously oxidized to Cys‐SO2H and Cys‐SOH, respectively, and αCys112‐SO2H is responsible for the catalytic activity solely or in combination with αCys114‐SOH.
A model for the sickle hemoglobin fiber using both mutation sites
Authors: Roufberg Adam , Ferrone Frank A.
Published: February 2009

The standard molecular model of the fiber of the sickle hemoglobin (Hbs: β6 Glu → Val) has been revised to allow both β6 mutation sites to participate in intermolecular contacts, rather than only one β6 site as previously thought, for four molecules per 14‐molecule fiber cross section. This structure accurately predicts the copolymerization of hybridized mixtures of HbS with HbA or HbC (β6 Glu → Lys), which could not be reconciled with prior models in which only half the β6 sites were required for assembly. This model suggests new contacts within the fiber and raises the question of whether these cross‐linked double strands could possess added stability important in such processes as nucleation.
Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor
Authors: Petersen Lars C. , Ård Per‐Ola Freskg , Persson Egon , Olsen Ole H. , Nielsen Lars S.
Published: February 2009

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn2+ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn2+ bind with high affinity to FVIIa outside the N‐terminal y‐carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ binding sites in the Glu210–Glu220 Ca2+ binding loop adjacent to the so‐called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn2+‐mediated allosteric down‐regulation of FVIIa's activity.
Conformational transitions and fibrillation mechanism of human calcitonin as studied by high‐resolution solid‐state 13C NMR
Authors: Kamihira Miya , Naito Akira , Tuzi Satoru , Saitô Hazime , Nosaka Atsuko Y.
Published: February 2009

Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high‐resolution solid‐state 13C NMR spectroscopy. In aqueous acetic acid solution (pH 3.3), a local α‐helical form is present around Gly10, whereas a random coil form is dominant as viewed from Phe22, Ala26, and Ala31 in the monomer form on the basis of the 13C chemical shifts. On the other hand, a local β‐sheet form as viewed from Gly10 and Phe22, and both β‐sheet and random coil as viewed from Ala26 and Ala31 were detected in the fibril at pH 3.3. The results indicate that conformational transitions from α‐helix to β‐sheet, and from random coil to β‐sheet forms occurred in the central and C‐terminus regions, respectively, during the fibril formation. The increased 13C resonance intensities of fibrils after a certain delay time suggests that the fibrillation can be explained by a two‐step reaction mechanism in which the first step is a homogeneous association to form a nucleus, and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a local β‐sheet conformation at the central region and exhibited a random coil at the C‐terminus region. Not only a hydrophobic interaction among the amphiphilic α‐helices, but also an electrostatic interaction between charged side chains can play an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel β‐sheets at pH 7.5 and a mixture of antiparallel and parallel β‐sheets at pH 3.3.
Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin—biotin system
Authors: Hyre David E. , Stayton Patrick S. , Trong Isolde Le , Freitag Stefanie , Stenkamp Ronald E.
Published: February 2009

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X‐ray crystallographic studies. The S45A mutant exhibits a 1,700‐fold greater disso‐ciation rate and 907‐fold lower equilibrium affinity for biotin relative to wild‐type streptavidin at 37dGC, indicating a crucial role in binding energetics. The crystal structure of the biotin‐bound mutant reveals only small changes from the wild‐type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium ΔG°, ΔH°, ΔS°, ΔCP°, and activation ΔG‡ of S45A at 37°C are ‐13.7 ± 0.1 kcal/mol, ‐21.1 ± 0.5 kcal/mol, ‐23.7 ± 1.8 cal/molK, ‐223 ± 12 cal/mol K, and 20.0 ± 2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off‐rate resolves the ΔH‡ and ΔS‡ of dissociation, 25.8 ± 1.2 kcal/mol and 18.7 ± 4.3 cal/mol K. The large increases of ΔH‡ and ΔS‡ in the mutant, relative to wild‐type, indicate that Ser45 could form a hydrogen bond with biotin in the wild‐type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45‐mediated hydrogen bond in the wild‐type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.
Conformational flexibility in the apolipoprotein E amino‐terminal domain structure determined from three new crystal forms: Implications for lipid binding
Authors: Segelke Brent W. , Forstner Michael , Knapp Mark , Trakhanov Sergei D. , Parkin Sean , Newhouse Yvonne M. , Bellamy Henry D. , Weisgraber Karl H. , Rupp Bernhard
Published: February 2009

An amino‐terminal fragment of human apolipoprotein E3 (residues 1‐165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P212121) crystal form of the amino‐terminal 22 kDa fragment of apolipoprotein E (residues 1‐191). A second orthorhombic crystal form (P212121 with cell dimensions differing from the first form) and a trigonal (P3121) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno‐methionine multiwavelength anomalous dispersion, and the structure of the second ortho‐rhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high‐quality electron‐density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four‐helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four‐helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.
Alternate modes of binding in two crystal structures of alkaline phosphatase‐inhibitor complexes
Authors: Holtz Kathleen M. , Kantrowitz Evan R. , Stec Boguslaw , Myers Jason K. , Antonelli Stephen M. , Widlanski Theodore S.
Published: February 2009

Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R‐values of 19.0 and 17.5%, respectively. Refinement of the AP‐inhibitor complexes was carried out using X‐PLOR. The final round of refinement was done using SHELXL‐97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X‐ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme‐inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C‐H…X.
Penicillopepsin‐JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to kcat
Authors: Stubbs Marlene , Ngo Kenny Q.P. , Cunningham Annie , Pai Emil F. , Hofmann Theo , Tu Guang‐Chou , Cao Qing‐Na , Ward Michael
Published: February 2009

The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin‐JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT‐pyrG1, was expressed in an aspartic proteinase‐free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin‐JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate‐enhancing effects of the presence of alanine residues in positions P3 and P2′ of substrates, are similar to those of penicillopepsin‐JT1, endothia‐pepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate‐enhancing effect was due to a hydrogen bond between the ‐NH‐of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C‐terminal to Asp215. Thr219 of penicillopepsin‐JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in kcat when a P3 residue was present in the substrate, which was similar to that of the wild‐type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the ‐NH‐ of P3 to the enzyme, the scissile peptide bond between P1 and P1′ becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.
Three‐dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors
Authors: Harel Michal , Kryger Gitay , Rosenberry Terrone L. , Mallender William D. , Lewis Terence , Fletcher Rodney J. , Guss J.Mitchell , Silman Israel , Sussman Joel L.
Published: February 2009

We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4‐tetrahydro‐N‐(phenylmethyl)‐9‐acridinamine and 1,2,3,4‐tetrahydro‐N‐(3‐iodophenyl‐methyl)‐9‐acridinamine—all three at 2.7 Å resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active‐site gorge, but some of the surface loops deviate by up to 8 Å from their position in the vertebrate structures, and the C‐terminal helix is shifted substantially. The active‐site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is ∼50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active‐site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three‐dimensional structures.