Protein Science current issue

[REVIEWS] Allostery and cooperativity revisited

Cui, Q., Karplus, M. — 2008-07-30

Although phenomenlogical models that account for cooperativity in allosteric systems date back to the early and mid-60's (e.g., the KNF and MWC models), there is resurgent interest in the topic due to the recent experimental and computational studies that attempted to reveal, at an atomistic level, how allostery actually works. In this review, using systems for which atomistic simulations have been carried out in our groups as examples, we describe the current understanding of allostery, how the mechanisms go beyond the classical MWC/Pauling-KNF descriptions, and point out that the "new view" of allostery, emphasizing "population shifts," is, in fact, an "old view." The presentation offers not only an up-to-date description of allostery from a theoretical/computational perspective, but also helps to resolve several outstanding issues concerning allostery.

[REVIEWS] Kinetic barriers and the role of topology in protein and RNA folding

Sosnick, T. R. — 2008-07-30

This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway.

[ACCELERATED COMMUNICATIONS] NMR insights into a megadalton-size protein self-assembly

Chugh, J., Sharma, S., Hosur, R. V. — 2008-07-30

Protein self-association is critical to many biological functions. However, atomic-level structural characterization of these assemblies has remained elusive. In this report we present insights into the mechanistic details of the process of self-association of the 136-residue GTPase effector domain (GED) of the endocytic protein dynamin into a megadalton-sized soluble mass. Our approach is based on NMR monitoring of regulated folding and association through Gdn-HCl titration. The results suggest the evolution of a sequence–self-association paradigm. Equally significantly, the study demonstrates an elegant bottom-up strategy that can render large protein self-assemblies accessible to NMR investigations that have remained difficult to date.

[ARTICLES] Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors

Abdiche, Y. N., Malashock, D. S., Pons, J. — 2008-07-30

We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.

[ARTICLES] The structure of an archaeal homodimeric ligase which has RNA circularization activity

2008-07-30

The genome of Pyrococcus abyssi contains two open reading frames encoding proteins which had been previously predicted to be DNA ligases, Pab2002 and Pab1020. We show that while the former is indeed a DNA ligase, Pab1020 had no effect on the substrate deoxyoligo-ribonucleotides tested. Instead, Pab1020 catalyzes the nucleotidylation of oligo-ribonucleotides in an ATP-dependent reaction, suggesting that it is an RNA ligase. We have solved the structure of Pab1020 in complex with the ATP analog AMPPNP by single-wavelength anomalous dispersion (SAD), elucidating a structure with high structural similarity to the catalytic domains of two RNA ligases from the bacteriophage T4. Additional carboxy-terminal domains are also present, and one of these mediates contacts with a second protomer, which is related by noncrystallographic symmetry, generating a homodimeric structure. These C-terminal domains are terminated by short domain swaps which themselves end within 5 Å of the active sites of the partner molecules. Additionally, we show that the protein is indeed capable of circularizing RNA molecules in an ATP-dependent reaction. These structural and biochemical results provide an insight into the potential physiological roles of Pab1020.

[ARTICLES] The structural basis of proteolytic activation of bovine glutamate dehydrogenase

Carrigan, J. B., Engel, P. C. — 2008-07-30

In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that came to light during those studies. We find that superactivation is caused by cleavage at the N terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in nontreated chymotrypsin may have led to the production of the larger 4–5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.

[ARTICLES] Computational study of the putative active form of protein Z (PZa): Sequence design and structural modeling

Chandrasekaran, V., Lee, C. J., Duke, R. E., Perera, L., Pedersen, L. G. — 2008-07-30

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.

[ARTICLES] Crystal structure and mutagenic analysis of GDOsp, a gentisate 1,2-dioxygenase from Silicibacter Pomeroyi

2008-07-30

Dioxygenases catalyze dioxygen incorporation into various organic compounds and play a key role in the complex degradation pathway of mono- and polycyclic aromatic and hetero-aromatic compounds. Here we report the crystal structure of gentisate 1,2-dioxygenase from Silicibacter pomeroyi (GDOsp) at a 2.8 Å resolution. The enzyme possessed a conserved three-dimensional structure of the bicupin family, forming a homotetramerization. However, each subunit of GDOsp unusually contained two ferrous centers that were located in its two homologous cupin domains, respectively. Further mutagenic analysis indicated that the enzyme activity of GDOsp depends on the microenvironment in both metal-binding sites. Moreover, homologous structural comparison and functional study on GDOsp variants unveiled a group of functionally essential residues and suggested that the active site of the enzyme is located in the amino-terminal domain, but could be influenced by changes in the carboxyl domain. Therefore, GDOsp may provide a working model for studying long-distance communication within a protein (or its complex).

[ARTICLES] Novel protein folds and their nonsequential structural analogs

Guerler, A., Knapp, E.-W. — 2008-07-30

Newly determined protein structures are classified to belong to a new fold, if the structures are sufficiently dissimilar from all other so far known protein structures. To analyze structural similarities of proteins, structure alignment tools are used. We demonstrate that the usage of nonsequential structure alignment tools, which neglect the polypeptide chain connectivity, can yield structure alignments with significant similarities between proteins of known three-dimensional structure and newly determined protein structures that possess a new fold. The recently introduced protein structure alignment tool, GANGSTA, is specialized to perform nonsequential alignments with proper assignment of the secondary structure types by focusing on helices and strands only. In the new version, GANGSTA+, the underlying algorithms were completely redesigned, yielding enhanced quality of structure alignments, offering alignment against a larger database of protein structures, and being more efficient. We applied DaliLite, TM-align, and GANGSTA+ on three protein crystal structures considered to be novel folds. Applying GANGSTA+ to these novel folds, we find proteins in the ASTRAL40 database, which possess significant structural similarities, albeit the alignments are nonsequential and in some cases involve secondary structure elements aligned in reverse orientation. A web server is available at http://agknapp.chemie.fu-berlin.de/gplus for pairwise alignment, visualization, and database comparison.

[ARTICLES] Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding

Parkkinen, T., Koivula, A., Vehmaanpera, J., Rouvinen, J. — 2008-07-30

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single-module, cellulose-degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello-oligomers: cellobiose (Glc₂), cellotriose (Glc₃), and cellotetraose (Glc₄), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β-sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from –5 to +2. In four cellotriose and one cellotetraose complex structures, the cello-oligosaccharide also spanned over the cleavage site (–1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello-oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (⁴C₁) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.

[ARTICLES] Tissue transglutaminase modulates {alpha}-synuclein oligomerization

2008-07-30

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated -synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant -synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective -synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all -synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal -synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal β-sheet-containing oligomers, the tTG/-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant -synuclein variants. We propose that tTG cross-linking imposes structural constraints on -synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

[ARTICLES] What is the role of the second "structural" NADP+-binding site in human glucose 6-phosphate dehydrogenase?

Wang, X.-T., Chan, T. F., Lam, V. M.S., Engel, P. C. — 2008-07-30

Human glucose 6-phosphate dehydrogenase, purified after overexpression in E. coli, was shown to contain one molecule/subunit of acid-extractable "structural" NADP⁺ and no NADPH. This tightly bound NADP⁺ was reduced by G6P, presumably following migration to the catalytic site. Gel-filtration yielded apoenzyme, devoid of bound NADP⁺ but, surprisingly, still fully active. M_r of the main component of "stripped" enzyme by gel filtration was ~100,000, suggesting a dimeric apoenzyme (subunit M_r = 59,000). Holoenzyme also contained tetramer molecules and, at high protein concentration, a dynamic equilibrium gave an apparent intermediate M_r of 150 kDa. Fluorescence titration of the stripped enzyme gave the K _d for structural NADP⁺ as 37 nM, 200-fold lower than for "catalytic" NADP⁺. Structural NADP⁺ quenches 91% of protein fluorescence. At 37°C, stripped enzyme, much less stable than holoenzyme, inactivated irreversibly within 2 d. Inactivation at 4°C was partially reversed at room temperature, especially with added NADP⁺. Apoenzyme was immediately active, without any visible lag, in rapid-reaction studies. Human G6PD thus forms active dimer without structural NADP⁺. Apparently, the true role of the second, tightly bound NADP⁺ is to secure long-term stability. This fits the clinical pattern, G6PD deficiency affecting the long-lived non-nucleate erythrocyte. The K _d values for two class I mutants, G488S and G488V, were 273 nM and 480 nM, respectively (seven- and 13-fold elevated), matching the structural prediction of weakened structural NADP⁺ binding, which would explain decreased stability and consequent disease. Preparation of native apoenzyme and measurement of K _d constant for structural NADP⁺ will now allow quantitative assessment of this defect in clinical G6PD mutations.

[ARTICLES] Crystal structure of a carbonyl reductase from Candida parapsilosis with anti-Prelog stereospecificity

2008-07-30

A novel short-chain (S)-1-phenyl-1,2-ethanediol dehydrogenase (SCR) from Candida parapsilosis exhibits coenzyme specificity for NADPH over NADH. It catalyzes an anti-Prelog type reaction to reduce 2-hydroxyacetophenone into (S)-1-phenyl-1,2-ethanediol. The coding gene was overexpressed in Escherichia coli and the purified protein was crystallized. The crystal structure of the apo-form was solved to 2.7 Å resolution. This protein forms a homo-tetramer with a broken 2-2-2 symmetry. The overall fold of each SCR subunit is similar to that of the known structures of other homologous alcohol dehydrogenases, although the latter usually form tetramers with perfect 2-2-2 symmetries. Additionally, in the apo-SCR structure, the entrance of the NADPH pocket is blocked by a surface loop. In order to understand the structure–function relationship of SCR, we carried out a number of mutagenesis–enzymatic analyses based on the new structural information. First, mutations of the putative catalytic Ser-Tyr-Lys triad confirmed their functional role. Second, truncation of an N-terminal 31-residue peptide indicated its role in oligomerization, but not in catalytic activity. Similarly, a V270D point mutation rendered the SCR as a dimer, rather than a tetramer, without affecting the enzymatic activity. Moreover, the S67D/H68D double-point mutation inside the coenzyme-binding pocket resulted in a nearly 10-fold increase and a 20-fold decrease in the k_cat /K_M value when NADH and NADPH were used as cofactors, respectively, with k_cat remaining essentially the same. This latter result provides a new example of a protein engineering approach to modify the coenzyme specificity in SCR and short-chain dehydrogenases/reductases in general.

[ARTICLES] Early events in protein folding: Is there something more than hydrophobic burst?

Camilloni, C., Sutto, L., Provasi, D., Tiana, G., Broglia, R. A. — 2008-07-30

The presence of native contacts in the denatured state of many proteins suggests that elements of the biologically active structure of these molecules are formed during the initial stage of the folding process. The rapidity with which these events take place makes it difficult to study them in vitro, but, by the same token, suitable for studies in silico. With the help of all-atom, explicit solvent, molecular dynamics simulations we have followed in time, starting from elongated structureless conformations, the early events in the folding of src-SH3 domain and of proteins G, L, and CI2. It is observed that within the first 50 ns two important events take place, essentially independent of each other: hydrophobic collapse and formation of a few selected native contacts. The same contacts are also found in simulations carried out in the presence of guanidinium chloride in order to reproduce the conditions used to characterize experimentally the denatured state and testify to the fact that these contacts are to be considered a resilient characterizing property of the denaturated state.

[ARTICLES] Hydrogen exchange of monomeric {alpha}-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Croke, R. L., Sallum, C. O., Watson, E., Watt, E. D., Alexandrescu, A. T. — 2008-07-30

Amide proton NMR signals from the N-terminal domain of monomeric -synuclein (S) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in -helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of S than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. C chemical shift data point to <10% residual -helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of S encapsulated within living Escherichia coli cells. Our finding that temperature effects on S NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that S remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of S with membranes and fibrils.

[ARTICLES] Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

2008-07-30

This paper reports that the acetylation of lysine -NH₃ ⁺ groups of -amylase—one of the most important hydrolytic enzymes used in industry—produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild-type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90°C in solutions containing 100-mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated -amylase toward inactivation is attributed to the increased net negative charge of -amylase that resulted from the acetylation of lysine ammonium groups (lysine -NH₃ ⁺ -> -NHCOCH₃). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial -amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.

[FOR THE RECORD] The polybasic region of Rho GTPases defines the cleavage by Yersinia enterocolitica outer protein T (YopT)

Fueller, F., Schmidt, G. — 2008-07-30

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins ( Yersinia outer proteins [Yops]), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell resulting in cell rounding. YopT is a cysteine protease that cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to inactivation. Small GTPases are modified by isoprenylation of the cysteine of the CAAX box, cleavage of the –AAX tripeptide, and methylation of the cysteine. We have shown that isoprenylation and the endoproteolytic cleavage of the tripeptide of Rho GTPases are essential for YopT-induced cleavage, whereas carboxyl methylation is not required. In the present study, we post-translationally modified RhoA, Rac, Cdc42, and several mutants in vitro and characterized the YopT-induced cleavage with recombinant YopT. We show that farnesylated RhoA is a preferred substrate of YopT compared with the geranylgeranylated GTPase. Geranylgeranylated RhoA, however, is the preferred substrate for YopT-catalyzed cleavage with a threefold faster turnover rate over Rac and Cdc42. Moreover, our data indicate that the composition of the polybasic region of the GTPases defines the specificity and efficiency of the YopT-induced cleavage, and that a space between the polybasic stretch of amino acids at the C terminus and the CAAX box enhances the turnover rate of YopT-catalyzed cleavage.