Protein and Peptide Letters - Volume 17, Issue 8, 2010

The notion that protein function relies on a precise 3D structure constitutes one of the central paradigms of biochemistry. According to this concept, a protein can perform its biological function(s) only after folding into a unique 3D structure, and all the information necessary for a protein to fold into this unique 3D structure (in a given environment) is encoded in the amino acid sequence. Only recently has the validity of this structure-function paradigm been seriously challenged, primarily through the wealth of counterexamples that have gradually accumulated over the past 20-25 years. These counterexamples demonstrated that many functional proteins or protein parts exist in an entirely or partly disordered state. Intrinsically disordered proteins (IDPs), also referred to as natively unfolded proteins, lack a unique, stable 3D structure in solution, existing instead as a dynamic ensemble of conformations and exerting their biological activity without a prerequisite stably folded structure. IDPs possess a distinct combination of a high content of charged residues and of a low content of hydrophobic residues that allows them to be distinguished from globular proteins. These peculiar sequence features have led to the development of various disorder predictors, which allowed an estimation of the occurrence of disorder in biological systems. These studies showed that the frequency and length of disordered regions increases with increasing organism's complexity. For example, long intrinsically disordered regions have been predicted to occur in 33% of eukaryotic proteins, with 12% of these latter being fully disordered. Furthermore, viruses and eukaryota were predicted to have ten times more conserved disorder (roughly 1%) than archaea and bacteria (0.1%). Beyond these computational studies, an increasing amount of experimental evidence has been gathered in the last decade pointing out the large abundance of intrinsic disorder within the living world: more than 523 proteins containing 1195 disordered regions have been annotated so far in the Disprot data base (http://www.disprot.org). Despite this large body of experimental evidence pointing out the abundance (and biological relevance) of disorder in the living world, the notion of a tight dependence of protein function on a precise 3D structure is still deeply anchored in many scientists' mind. The reasons for this lack of awareness or even “resistance” to the concept of protein intrinsic disorder are multiple. First, the growing numbers of protein structures determined by X-ray crystallography and by NMR in the last three decades has shifted the attention of scientists away from the numerous examples of IDPs. Second, IDPs have been long unnoticed because researchers encountering examples of structural disorder mainly ascribed them to errors and artifacts and, as such, purged them from papers and reports. Third, structural disorder is hard to conceive and classify. Fourth, IDPs have been neglected because of the perception that a limited amount of mechanistic data could be derived from their study. Yet, the evidence that IDPs exist both in vitro and in vivo is compelling and justifies considering them as a separate class within the protein realm. Many IDPs undergo a disorder-to-order transition upon binding to their physiological partner(s), a process termed induced folding. IDPs bind to their target(s) through “molecular recognition elements” (MoREs) or “molecular recognition features” (MoRFs). MoRFs are interaction-prone short segments with an increased foldability, which are embedded within long disordered regions and which become ordered upon binding to a specific partner. The conformation of MoRFs in isolation can be either disordered or partially preformed, thus reflecting an inherent conformational preference. In this latter case, a transiently populated folded state would exist even in the absence of the partner, thus implying that the folding induced by the partner would rely (at least partly) on conformer selection (i.e. selection by the partner of a pre-existing conformation) rather than on a “fly-casting” mechanism. It has been proposed that the restriction in the conformational space of MoRFs in the unbound state could reduce the entropic cost of binding thereby enhancing affinity. IDPs can bind their target(s) with a high extent of conformational polymorphism, with binding generally involving larger normalized interface areas than those found between rigid partners, with protein interfaces being enriched in hydrophobic residues. Thus, protein-protein interactions established by IDPs rely more on hydrophobic-hydrophobic than on polar-polar contacts....

Many proteins or their regions are disordered in their native, biologically active states. Bioinformatics has revealed that these proteins/regions are highly abundant in different proteomes and carry out mostly regulatory functions related to molecular recognition, signal transduction, protein-protein, and protein-nucleic acid interactions. Viruses, these “organisms at the edge of life”, have uniquely evolved to be highly adaptive for fast change in their biological and physical environment. To sustain these fast environmental changes, viral proteins elaborated multiple measures, from relatively low van der Waals contact densities, to inclusion of a large fraction of residues that are not arranged in well-defined secondary structural elements, to heavy use of short disordered regions, and to high resistance to mutations. On the other hand, viral proteins are rich in intrinsic disorder. Some of the intrinsically disordered regions are heavily used in the functioning of viral proteins. Others likely have evolved to help viruses accommodate to their hostile habitats. Still others evolved to help viruses in managing their economic usage of genetic material via alternative splicing, overlapping genes, and anti-sense transcription. In this review, we focus on structural peculiarities of viral proteins and on the role of intrinsic disorder in their functions.

Intrinsically disordered regions of significant length are present throughout eukaryotic genomes, and are particularly prevalent in viral proteins. Due to their inherent flexibility, these proteins inhabit a conformational landscape that is too complex to be described by classical structural biology. The elucidation of the role that conformational flexibility plays in molecular function will redefine our understanding of the molecular basis of biological function, and the development of appropriate technology to achieve this aim remains one of the major challenges for the future of structural biology. NMR is the technique of choice for studying intrinsically disordered proteins, providing information about structure, flexibility and interactions at atomic resolution even in completely disordered proteins. In particular residual dipolar couplings (RDCs) are sensitive and powerful tools for determining local and long-range structural behaviour in flexible proteins. Here we describe recent applications of the use of RDCs to quantitatively describe the level of local structure in intrinsically disordered proteins involved in replication and transcription in Sendai virus.

In this review, we summarize the main experimental data showing the abundance of structural disorder within the measles virus (MeV) nucleoprotein (N) and phosphoprotein (P), and focus on the molecular mechanisms governing the disorder-to-order transition of the intrinsically disordered C-terminal domain of MeV N (NTAIL) upon binding to the C-terminal X domain of P (XD). The functional implications of structural disorder are discussed in light of the ability of disordered regions to establish a complex molecular partnership, thereby leading to a variety of biological effects, including tethering of the polymerase complex onto the nucleocapsid template, stimulation of viral transcription and replication, and virus assembly. We also discuss the ability of NTAIL to establish interactions with additional cellular co-factors, including the major inducible heat shock protein, which can modulate the strength of the NTAIL-XD interaction. Taking into account the promiscuity that typifies disordered regions, we propose that the main functional advantage of the abundance of disorder within viruses would reside in pleiotropy and genetic compaction, where a single gene would encode a single (regulatory) protein product able to establish multiple interactions via its disordered regions, and hence to exert multiple concomitant biological effects.

Rhabdoviridae are single stranded negative sense RNA viruses. The viral RNA condensed by the nucleoprotein (N), the phosphoprotein (P) and the large subunit (L) of the RNA-dependent RNA polymerase are the viral components of the transcription/replication machineries. Both P and N contain intrinsically disordered regions (IDRs) that play different roles in the virus life cycle. Here, we describe the modular organization of P based on recent structural, biophysical and bioinformatics data. We show how flexible loops in N participate in the attachment of P to the N-RNA template by an induced- fit mechanism. Finally, we discuss the roles of IDRs in the mechanism of replication/transcription, and propose a new model for the interaction of the L subunit with its N-RNA template.

The HIV-1 Vif protein (192 residues) is required for HIV-1 infection of many target cells. Vif overcomes the anti-viral cellular defense by antagonizing the cellular cytosine deaminase APOBEC-3G through impairing APOBEC-3G production, inhibiting its enzymatic activity and targeting it for degradation. Vif interacts with several viral and cellular molecules, particularly via its C-terminal domain (residues 100-192). The structure of full-length Vif has not yet been determined. The structure of Vif and its domains was studied using computational and experimental methods. Computational predictions resulted in two suggested homology models for the full length protein. Experimental studies have shown that the Vif C-terminal domain is mainly unstructured. Residues 108-139 have mainly random coil conformation in the unbound state. This region includes an HCCH Zn2+-binding motif that also mediates Vif binding to Cul5, a protein in the E3 ubiquitin ligase complex. The C-terminal domain residues 141-192, which mediate interactions with both ElonginC and Cul5, are intrinsically disordered. This region also includes several phosphorylation sites and regions associated with the ability of Vif to undergo self-oligomerization. The unstructured nature of these regions enables them to interact with several ligands, and probably adopt various conformations as is typical for intrinsically disordered proteins. This was demonstrated by a conformational change induced by Zn2+ binding to the HCCH motif and a conformational change that the C-terminal domain underwent in the presence of dodecylphosphocholine. The only available crystal structure of Vif includes residues 140-155, which are helical when bound to the ElonginBC complex. Overall, empirical structures, predictions and other experimental data for Vif did not always indicate the same degree or type of structure for any given region. This ambiguity is likely to be the tenet of structurally unfolded proteins, which have the propensity to adopt a multitude of biologically relevant and active conformations.

The type 1 Human Immunodeficiency Virus transcriptional regulator Tat is a small RNA-binding protein essential for viral gene expression and replication. The protein binds to a large number of proteins within infected cells and non-infected cells, and has been demonstrated to impact a wide variety of cellular activities. Early circular dichroism studies showed a lack of regular secondary structure in the protein whereas proton NMR studies suggested several different conformations. Multinuclear NMR structure and dynamics analysis indicates that the reduced protein is intrinsically disordered with a predominantly extended conformation at pH 4. Multiple resonances for several atoms suggest the existence of multiple local conformers in rapid equilibrium. An X-ray diffraction structure of equine Tat, in a complex with its cognate RNA and cyclin T1, supports this conclusion. Intrinsic disorder explains the protein's capacity to interact with multiple partners and effect multiple biological functions; the large buried surface in the X-ray diffraction structure illustrates how a disordered protein can have a high affinity and high specificity for its partners and how disordered Tat assembles a protein complex to enhance transcription elongation.

We present here our current understanding of the NS5A-D2 domain of the hepatitis C virus. Whereas this protein domain is globally unstructured as assessed by macroscopic techniques such as size exclusion chromatography, circular dichroism and homonuclear NMR spectroscopy, high resolution triple resonance spectroscopy allows the identification of a small region of residual structure. This region corresponds moreover to the most conserved sequence over the different genotypes of the virus, underscoring its functional importance. We show that it forms an anchoring point for the host cell cyclophilin prolyl cis/trans isomerase, providing a molecular basis for the use of cyclophilin inhibitors in an antiviral strategy.

Hepatitis C virus and related viruses in the Flaviviridae family (such as dengue virus, yellow fever virus or West Nile virus) are amongst the most important human pathogens, causing substantial morbidity and mortality worldwide. The production of viral progeny in Flaviviridae is orchestrated by the small, multifunctional core protein, which coats and condenses the viral genomic RNA during Nucleocapsid formation. In addition to their structural role, mounting experimental evidence links core proteins to viral persistence and pathogenesis, by virtue of their promiscuous interactions with host cell factors. In this review, we summarize the present knowledge about the structure of Flaviviridae core proteins and discuss the importance of flexible, intrinsically unstructured protein regions in viral assembly and hub formation in the virus-host protein-protein interaction network (infection network).

The human tyrosinase ectodomain has been expressed in Escherichia coli as a soluble form and purified by immobilized metal affinity column chromatography. The ectodomain exhibited tyrosinase activities toward the hydroxylation and oxidation reactions. Biochemical properties of the ectodomain appeared to be distinct from those of the human tyrosinase, although common features were retained.

There is no protective vaccine or effective drug against hepatitis C virus (HCV). Sustained virological response to INF/ribavirin treatment regimen has an efficiency of about 50%. Many patients worldwide have used traditional medicines and herbal medicine in particular. A laccase has been purified from oyster mushroom (Pleurotus ostreatus) to homogeneity by DEAE Affi-gel blue gel, CM-Sephadex G-50 and Sephadex G-100. The molecular weight of the laccase was about 58 kDa in SDS-PAGE. The optimum pH and temperature of the laccase activity were pH 4.0 and 60°C, respectively. The activity of the enzyme increased steadily from 20 to 40°C, then very slowly from 40°to 60°C, while the enzyme activity decreased to 9% at 90°C. The activity of the laccase changed gradually over the pH range 2.0-4.0. However, the enzyme activity was totally abrogated at the pH 8 and above. Incubation of peripheral blood cells PBCs and hepatoma HepG2 cells with laccase which were then infected with HCV did not protect the cells from HCV attack and entry, while direct interaction between HCV and the laccase at the concentrations of 2.0 and 2.5 mg/ml led to a complete inhibition of virus entry after seven days of incubation. Meantime, the laccase at the concentrations of 1.0 and 1.5 mg/ml did not display any blocking activity. The potential activity of the laccase on intracellular HCV replication in infected HepG2 cells has been examined. The laccase was capable of inhibiting HCV replication at the concentrations of 1.25 and 1.5 mg/ml after first dose of treatment for four days and at the concentrations of 0.75, 1.0, 1.25 and 1.5 mg/ml after the second dose of treatment for another four days.

A 66-kDa laccase, with an N-terminal sequence different from those of other mushroom laccases, was purified from fresh fruiting bodies of the edible mushroom Pleurotus cornucopiae by using affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Mono Q and gel filtration on Superdex 75. The procedure resulted in a 16-fold purification and a specific enzyme activity of 17.3 U mg-1. The optimum pH and temperature for the purified laccase were pH 4 and 40°C, respectively. This laccase inhibited proliferation of murine leukemia cell line L1210 and human hepatoma cell line HepG2, and reduced the activity of HIV-1 reverse transcriptase with an IC50 of 22μM. There was neither mitogenic activity toward mouse splenocytes, nor hemagglutinating/hemolytic activity toward rabbit erythrocytes. This study yielded information about the potentially exploitable activities of P. cornucopiae laccase.

Mouse liver cytosolic malate dehydrogenase was separated by non-denaturing two-dimensional electrophoresis and identified. Furthermore, the activity of the enzyme was preserved even after separation, electroblotting onto a membrane and staining with Ponceau S in acidic buffer solution (pH 5.1). Using the membrane-immobilized enzyme, the malic acid content was estimated by measuring absorbance changes due to the conversion of nicotinamide adenine dinucleotide (NAD) to NADH. These results indicate that enzyme reactors can be systematically produced after purification, immobilization and staining with Ponceau S.

D-Alanine:D-Alanine ligase (DDl) catalyzes the formation of D-Alanine:D-Alanine dipeptide and is an essential enzyme in bacterial cell wall biosynthesis. This enzyme does not have a human ortholog, making it an attractive target for developing new antibiotic drugs. We determined the crystal structure at 2.23 Å resolution of DDl from Streptococcus mutans (SmDDl), the principal aetiological agent of human dental caries. This structure reveals that SmDDl is a dimer and has a disordered ω-loop region.

The protective effects of four osmolytes (trehalose, dimethysulfoxide, glycine and proline) on the conformational stability and aggregation of guanidine-denatured yeast alcohol dehydrogenase (YADH) have been investigated in this paper. The results show that the four osmolytes protect YADH against unfolding and inactivation by reducing ki (inactivation rate constants), increasing ΔΔGi (transition free energy changes at 25°C), increasing Cm (value for the midpoint of denaturation) and decreasing its ANS-binding fluorescence intensity. Furthermore, these osmolytes can prevent YADH aggregation in a concentration-dependent manner during YADH refolding.