Brucella NpeA is a secreted Type IV effector containing an N-WASP-binding short linear motif that promotes niche formation.

The modulation of actin polymerization is a common theme among microbial pathogens. Even though microorganisms show a wide repertoire of strategies to subvert the activity of actin, most of them converge in the ones that activate nucleating factors, such as the Arp2/3 complex. Brucella spp. are intracellular pathogens capable of establishing chronic infections in their hosts. The ability to subvert the host cell response is dependent on the capacity of the bacterium to attach, invade, avoid degradation in the phagocytic compartment, replicate in an endoplasmic reticulum-derived compartment and egress. Even though a significant number of mechanisms deployed by Brucella in these different phases have been identified and characterized, none of them have been described to target actin as a cellular component. In this manuscript, we describe the identification of a novel virulence factor (NpeA) that promotes niche formation. NpeA harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP and stabilizes the autoinhibited state. Our results show that NpeA is secreted in a Type IV secretion system-dependent manner and that deletion of the gene diminishes the intracellular replication capacity of the bacterium. In vitro and ex vivo experiments demonstrate that NpeA binds N-WASP and that the short linear motif is required for the biological activity of the protein.IMPORTANCEThe modulation of actin-binding effectors that regulate the activity of this fundamental cellular protein is a common theme among bacterial pathogens. The neural Wiskott-Aldrich syndrome protein (N-WASP) is a protein that several pathogens target to hijack actin dynamics. The highly adapted intracellular bacterium Brucella has evolved a wide repertoire of virulence factors that modulate many activities of the host cell to establish successful intracellular replication niches, but, to date, no effector proteins have been implicated in the modulation of actin dynamics. We present here the identification of a virulence factor that harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP stabilizing its autoinhibited state. We demonstrate that this protein is a Type IV secretion effector that targets N-WASP-promoting intracellular survival and niche formation.

A disordered region retains the full protease inhibitor activity and the capacity to induce CD8+ T cells in vivo of the oral vaccine adjuvant U-Omp19.

U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.

Conformational buffering underlies functional selection in intrinsically disordered protein regions.

Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.

A Novel Bacterial Protease Inhibitor Adjuvant in RBD-Based COVID-19 Vaccine Formulations Containing Alum Increases Neutralizing Antibodies, Specific Germinal Center B Cells and Confers Protection Against SARS-CoV-2 Infection in Mice.

In this work, we evaluated recombinant receptor binding domain (RBD)-based vaccine formulation prototypes with potential for further clinical development. We assessed different formulations containing RBD plus alum, AddaS03, AddaVax, or the combination of alum and U-Omp19: a novel Brucella spp. protease inhibitor vaccine adjuvant. Results show that the vaccine formulation composed of U-Omp19 and alum as adjuvants has a better performance: it significantly increased mucosal and systemic neutralizing antibodies in comparison to antigen plus alum, AddaVax, or AddaS03. Antibodies induced with the formulation containing U-Omp19 and alum not only increased their neutralization capacity against the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants with similar potency. Furthermore, the addition of U-Omp19 to alum vaccine formulation increased the frequency of RBD-specific geminal center B cells and plasmablasts. Additionally, U-Omp19+alum formulation induced RBD-specific Th1 and CD8+ T-cell responses in spleens and lungs. Finally, this vaccine formulation conferred protection against an intranasal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge of K18-hACE2 mice.

Interplay between sequence, structure and linear motifs in the adenovirus E1A hub protein.

E1A is the main transforming protein in mastadenoviruses. This work uses bioinformatics to extrapolate experimental knowledge from Human adenovirus serotype 5 and 12 E1A proteins to all known serotypes. A conserved domain architecture with a high degree of intrinsic disorder acts as a scaffold for multiple linear motifs with variable occurrence mediating the interaction with over fifty host proteins. While linear motifs contribute strongly to sequence conservation within intrinsically disordered E1A regions, motif repertoires can deviate significantly from those found in prototypical serotypes. Close to one hundred predicted residue-residue contacts suggest the presence of stable structure in the CR3 domain and of specific conformational ensembles involving both short- and long-range intramolecular interactions. Our computational results suggest that E1A sequence conservation and co-evolution reflect the evolutionary pressure to maintain a mainly disordered, yet non-random conformation harboring a high number of binding motifs that mediate viral hijacking of the cell machinery.

Cooperative RNA Recognition by a Viral Transcription Antiterminator.

RNA transcription of mononegavirales decreases gradually from the 3' leader promoter toward the 5' end of the genome, due to a decay in polymerase processivity. In the respiratory syncytial virus and metapneumovirus, the M2-1 protein ensures transcription anti-termination. Despite being a homotetramer, respiratory syncytial virus M2-1 binds two molecules of RNA of 13mer or longer per tetramer, and temperature-sensitive secondary structure in the RNA ligand is unfolded by stoichiometric interaction with M2-1. Fine quantitative analysis shows positive cooperativity, indicative of conformational asymmetry in the tetramer. RNA binds to M2-1 through a fast bimolecular association followed by slow rearrangements corresponding to an induced-fit mechanism, providing a sequential description of the time events of cooperativity. The first binding event of half of the RNA molecule to one of the sites increases the affinity of the second binding event on the adjacent contacting protomer by 15-fold, product of increased effective concentration caused by the entropic link. This mechanism allows for high-affinity binding with an otherwise relaxed sequence specificity, and instead suggests a yet undefined structural recognition signature in the RNA for modulating gene transcription. This work provides a basis for an essential event for understanding transcription antitermination in pneumoviruses and its counterpart Ebola virus VP30.

Short linear motif core and flanking regions modulate retinoblastoma protein binding affinity and specificity.

Pocket proteins retinoblastoma (pRb), p107 and p130 are negative regulators of cellular proliferation and multifunctional proteins regulating development, differentiation and chromatin structure. The retinoblastoma protein is a potent tumor suppressor mutated in a wide range of human cancers, and oncogenic viruses often interfere with cell cycle regulation by inactivating pRb. The LxCxE and pRb AB groove short linear motifs (SLiMs) are key to many pocket protein mediated interactions including host and viral partners. A review of available experimental evidence reveals that several core residues composing each motif instance are determinants for binding. In the LxCxE motif, a fourth hydrophobic position that might allow variable spacing is required for binding. In both motifs, flanking regions including charged stretches and phosphorylation sites can fine-tune the binding affinity and specificity of pocket protein SLiM-mediated interactions. Flanking regions can modulate pocket protein binding specificity, or tune the high affinity interactions of viral proteins that hijack the pRb network. The location of SLiMs within intrinsically disordered regions allows faster evolutionary rates that enable viruses to acquire a functional variant of the core motif by convergent evolution, and subsequently test numerous combinations of flanking regions towards maximizing interaction specificity and affinity. This knowledge can guide future efforts directed at the design of peptide-based compounds that can target pocket proteins to regulate the G1/S cell cycle checkpoint or impair viral mediated pRb inactivation.

Hidden Structural Codes in Protein Intrinsic Disorder.

Intrinsic disorder is a major structural category in biology, accounting for more than 30% of coding regions across the domains of life, yet consists of conformational ensembles in equilibrium, a major challenge in protein chemistry. Anciently evolved papillomavirus genomes constitute an unparalleled case for sequence to structure-function correlation in cases in which there are no folded structures. E7, the major transforming oncoprotein of human papillomaviruses, is a paradigmatic example among the intrinsically disordered proteins. Analysis of a large number of sequences of the same viral protein allowed for the identification of a handful of residues with absolute conservation, scattered along the sequence of its N-terminal intrinsically disordered domain, which intriguingly are mostly leucine residues. Mutation of these led to a pronounced increase in both α-helix and ß-sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics, and uncovers the existence of local structural elements that oppose canonical folding. These folding relays suggest the existence of yet undefined hidden structural codes behind intrinsic disorder in this model protein. Thus, evolution pinpoints conformational hot spots that could have not been identified by direct experimental methods for analyzing or perturbing the equilibrium of an intrinsically disordered protein ensemble.

Naturally occurring fluorescence in frogs.

Fluorescence, the absorption of short-wavelength electromagnetic radiation reemitted at longer wavelengths, has been suggested to play several biological roles in metazoans. This phenomenon is uncommon in tetrapods, being restricted mostly to parrots and marine turtles. We report fluorescence in amphibians, in the tree frog Hypsiboas punctatus, showing that fluorescence in living frogs is produced by a combination of lymph and glandular emission, with pigmentary cell filtering in the skin. The chemical origin of fluorescence was traced to a class of fluorescent compounds derived from dihydroisoquinolinone, here named hyloins. We show that fluorescence contributes 18-29% of the total emerging light under twilight and nocturnal scenarios, largely enhancing brightness of the individuals and matching the sensitivity of night vision in amphibians. These results introduce an unprecedented source of pigmentation in amphibians and highlight the potential relevance of fluorescence in visual perception in terrestrial environments.

Intrinsic Disorder to Order Transitions in the Scaffold Phosphoprotein P from the Respiratory Syncytial Virus RNA Polymerase Complex.

Intrinsic disorder is at the center of biochemical regulation and is particularly overrepresented among the often multifunctional viral proteins. Replication and transcription of the respiratory syncytial virus (RSV) relies on a RNA polymerase complex with a phosphoprotein cofactor P as the structural scaffold, which consists of a four-helix bundle tetramerization domain flanked by two domains predicted to be intrinsically disordered. Because intrinsic disorder cannot be reduced to a defined atomic structure, we tackled the experimental dissection of the disorder-order transitions of P by a domain fragmentation approach. P remains as a tetramer above 70 °C but shows a pronounced reversible secondary structure transition between 10 and 60 °C. While the N-terminal module behaves as a random coil-like IDP in a manner independent of tetramerization, the isolated C-terminal module displays a cooperative and reversible metastable transition. When linked to the tetramerization domain, the C-terminal module becomes markedly more structured and stable, with strong ANS binding. Therefore, the tertiary structure in the C-terminal module is not compact, conferring "late" molten globule-like IDP properties, stabilized by interactions favored by tetramerization. The presence of a folded structure highly sensitive to temperature, reversibly and almost instantly formed and broken, suggests a temperature sensing activity. The marginal stability allows for exposure of protein binding sites, offering a thermodynamic and kinetic fine-tuning in order-disorder transitions, essential for the assembly and function of the RSV RNA polymerase complex.

Conformational Heterogeneity Determined by Folding and Oligomer Assembly Routes of the Interferon Response Inhibitor NS1 Protein, Unique to Human Respiratory Syncytial Virus.

The nonstructural NS1 protein is an essential virulence factor of the human respiratory syncytial virus, with a predominant role in the inhibition of the host antiviral innate immune response. This inhibition is mediated by multiple protein-protein interactions and involves the formation of large oligomeric complexes. There is neither a structure nor sequence or functional homologues of this protein, which points to a distinctive mechanism for blocking the interferon response among viruses. The NS1 native monomer follows a simple unfolding kinetics via a nativelike transition state ensemble, with a half-life of 45 min, in agreement with a highly stable core structure at equilibrium. Refolding is a complex process that involves several slowly interconverting species compatible with proline isomerization. However, an ultrafast folding event with a half-life of 0.2 ms is indicative of a highly folding compatible species within the unfolded state ensemble. On the other hand, the oligomeric assembly route from the native monomer, which does not involve unfolding, shows a monodisperse and irreversible end-point species triggered by a mild temperature change, with half-lives of 160 and 26 min at 37 and 47 °C, respectively, and at a low protein concentration (10 µM). A large secondary structure change into ß-sheet structure and the formation of a dimeric nucleus precede polymerization by the sequential addition of monomers at the surprisingly low rate of one monomer every 34 s. The polymerization phase is followed by the binding to thioflavin-T indicative of amyloid-like, albeit soluble, repetitive ß-sheet quaternary structure. The overall process is reversible only up until ~8 min, a time window in which most of the secondary structure change takes place. NS1's multiple binding activities must be accommodated in a few binding interfaces at most, something to be considered remarkable given its small size (15 kDa). Thus, conformational heterogeneity, and in particular oligomer formation, may provide a means of expand its binding repertoire. These equilibria will be determined by variables such as macromolecular crowding, protein-protein interactions, expression levels, turnover, or specific subcellular localization. The irreversible and quasi-spontaneous nature of the oligomer assembly, together with the fact that NS1 is the most abundant viral protein in infected cells, makes its accumulation highly conceivable under conditions compatible with the cellular milieu. The implications of NS1 oligomers in the viral life cycle and the inhibition of host innate immune response remain to be determined.

Cysteine-rich positions outside the structural zinc motif of human papillomavirus E7 provide conformational modulation and suggest functional redox roles.

The E7 protein from high-risk human papillomavirus is essential for cell transformation in cervical, oropharyngeal, and other HPV-related cancers, mainly through the inactivation of the retinoblastoma (Rb) tumor suppressor. Its high cysteine content (~7%) and the observation that HPV-transformed cells are under oxidative stress prompted us to investigate the redox properties of the HPV16 E7 protein under biologically compatible oxidative conditions. The seven cysteines in HPV16 E7 remain reduced in conditions resembling the basal reduced state of a cell. However, under oxidative stress, a stable disulfide bridge forms between cysteines 59 and 68. Residue 59 has a protective effect on the other cysteines, and its mutation leads to an overall increase in the oxidation propensity of E7, including cysteine 24 central to the Rb binding motif. Gluthationylation of Cys 24 abolishes Rb binding, which is reversibly recovered upon reduction. Cysteines 59 and 68 are located 18.6 Å apart, and the formation of the disulfide bridge leads to a large structural rearrangement while retaining strong Zn association. These conformational and covalent changes are fully reversible upon restoration of the reductive environment. In addition, this is the first evidence of an interaction between the N-terminal intrinsically disordered and the C-terminal globular domains, known to be highly and separately conserved among human papillomaviruses. The significant conservation of such noncanonical cysteines in HPV E7 proteins leads us to propose a functional redox activity. Such an activity adds to the previously discovered chaperone activity of E7 and supports the picture of a moonlighting pathological role of this paradigmatic viral oncoprotein.

Conformational dissection of a viral intrinsically disordered domain involved in cellular transformation.

Intrinsic disorder is abundant in viral genomes and provides conformational plasticity to its protein products. In order to gain insight into its structure-function relationships, we carried out a comprehensive analysis of structural propensities within the intrinsically disordered N-terminal domain from the human papillomavirus type-16 E7 oncoprotein (E7N). Two E7N segments located within the conserved CR1 and CR2 regions present transient α-helix structure. The helix in the CR1 region spans residues L8 to L13 and overlaps with the E2F mimic linear motif. The second helix, located within the highly acidic CR2 region, presents a pH-dependent structural transition. At neutral pH the helix spans residues P17 to N29, which include the retinoblastoma tumor suppressor LxCxE binding motif (residues 21-29), while the acidic CKII-PEST region spanning residues E33 to I38 populates polyproline type II (PII) structure. At pH 5.0, the CR2 helix propagates up to residue I38 at the expense of loss of PII due to charge neutralization of acidic residues. Using truncated forms of HPV-16 E7, we confirmed that pH-induced changes in α-helix content are governed by the intrinsically disordered E7N domain. Interestingly, while at both pH the region encompassing the LxCxE motif adopts α-helical structure, the isolated 21-29 fragment including this stretch is unable to populate an α-helix even at high TFE concentrations. Thus, the E7N domain can populate dynamic but discrete structural ensembles by sampling α-helix-coil-PII-ß-sheet structures. This high plasticity may modulate the exposure of linear binding motifs responsible for its multi-target binding properties, leading to interference with key cell signaling pathways and eventually to cellular transformation by the virus.

Folding of a cyclin box: linking multitarget binding to marginal stability, oligomerization, and aggregation of the retinoblastoma tumor suppressor AB pocket domain.

The retinoblastoma tumor suppressor (Rb) controls the proliferation, differentiation, and survival of cells in most eukaryotes with a role in the fate of stem cells. Its inactivation by mutation or oncogenic viruses is required for cellular transformation and eventually carcinogenesis. The high conservation of the Rb cyclin fold prompted us to investigate the link between conformational stability and ligand binding properties of the RbAB pocket domain. RbAB unfolding presents a three-state transition involving cooperative secondary and tertiary structure changes and a partially folded intermediate that can oligomerize. The first transition corresponds to unfolding of the metastable B subdomain containing the binding site for the LXCXE motif present in cellular and viral targets, and the second transition corresponds to the stable A subdomain. The low thermodynamic stability of RbAB translates into a propensity to rapidly oligomerize and aggregate at 37 °C (T50 = 28 min) that is suppressed by human papillomavirus E7 and E2F peptide ligands, suggesting that Rb is likely stabilized in vivo through binding to target proteins. We propose that marginal stability and associated oligomerization may be conserved for function as a "hub" protein, allowing the formation of multiprotein complexes, which could constitute a robust mechanism to retain its cell cycle regulatory role throughout evolution. Decreased stability and oligomerization are shared with the p53 tumor suppressor, suggesting a link between folding and function in these two essential cell regulators that are inactivated in most cancers and operate within multitarget signaling pathways.

Minute time scale prolyl isomerization governs antibody recognition of an intrinsically disordered immunodominant epitope.

Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a "hinge" region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t(1/2)∼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 10(7) M(-1) s(-1), a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with K(D) = 1.2 × 10(-7) M is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be "locked" in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation.

Sequence evolution of the intrinsically disordered and globular domains of a model viral oncoprotein.

In the present work, we have used the papillomavirus E7 oncoprotein to pursue structure-function and evolutionary studies that take into account intrinsic disorder and the conformational diversity of globular domains. The intrinsically disordered (E7N) and globular (E7C) domains of E7 show similar degrees of conservation and co-evolution. We found that E7N can be described in terms of conserved and coevolving linear motifs separated by variable linkers, while sequence evolution of E7C is compatible with the known homodimeric structure yet suggests other activities for the domain. Within E7N, inter-residue relationships such as residue co-evolution and restricted intermotif distances map functional coupling and co-occurrence of linear motifs that evolve in a coordinate manner. Within E7C, additional cysteine residues proximal to the zinc-binding site may allow redox regulation of E7 function. Moreover, we describe a conserved binding site for disordered domains on the surface of E7C and suggest a putative target linear motif. Both homodimerization and peptide binding activities of E7C are also present in the distantly related host PHD domains, showing that these two proteins share not only structural homology but also functional similarities, and strengthening the view that they evolved from a common ancestor. Finally, we integrate the multiple activities and conformations of E7 into a hierarchy of structure-function relationships.

Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains.

Circular dichroism (CD) spectroscopy is a simple and powerful technique, which allows for the assessment of the conformational properties of a protein or protein domain. Intrinsically disordered proteins (IDPs), as discussed throughout this series, differ from random coil polypeptides in that different regions present specific conformational preferences, exhibiting dynamic secondary structure content [1]. These dynamic secondary structure elements can be stabilized or perturbed by different chemical (solvent, ionic strength, pH) or physical (temperature) agents, by posttranslational modifications, and by ligands. This information is important for defining ID nature. As IDPs present dynamic conformations, circular dichroism measurements (and other approaches as well) should be carried out not as single spectra performed in unique conditions, but instead changing the chemical conditions and observing the behavior, as part of the determination of the ID nature.In this chapter, we present the basic methodology for performing Far-UV CD measurements on a protein of interest and for identifying and characterizing intrinsically disordered regions, and several protocols for the analysis of residual secondary structure present in the protein under study. These techniques are straightforward to perform; they require minimal training and can be preliminary to more complex methodologies such as NMR.

Evolution of linear motifs within the papillomavirus E7 oncoprotein.

Many protein functions can be traced to linear sequence motifs of less than five residues, which are often found within intrinsically disordered domains. In spite of their prevalence, their role in protein evolution is only beginning to be understood. The study of papillomaviruses has provided many insights on the evolution of protein structure and function. We have chosen the papillomavirus E7 oncoprotein as a model system for the evolution of functional linear motifs. The multiple functions of E7 proteins from paradigmatic papillomavirus types can be explained to a large extent in terms of five linear motifs within the intrinsically disordered N-terminal domain and two linear motifs within the globular homodimeric C-terminal domain. We examined the motif inventory of E7 proteins from over 200 known papillomavirus types and found that the motifs reported for paradigmatic papillomavirus types are absent from many uncharacterized E7 proteins. Several motif pairs occur more often than expected, suggesting that linear motifs may evolve and function in a cooperative manner. The E7 linear motifs have appeared or disappeared multiple times during papillomavirus evolution, confirming the evolutionary plasticity of short functional sequences. Four of the motifs appeared several times during papillomavirus evolution, providing direct evidence for convergent evolution. Interestingly, the evolution pattern of a motif is independent of its location in a globular or disordered domain. The correlation between the presence of some motifs and virus host specificity and tissue tropism suggests that linear motifs play a role in the adaptive evolution of papillomaviruses.

The respiratory syncytial virus transcription antiterminator M(2-1) is a highly stable, zinc binding tetramer with strong pH-dependent dissociation and a monomeric unfolding intermediate.

The human respiratory syncytial virus M(2-1) transcription antiterminator is an essential elongation factor required by the RNA polymerase for effective transcription beyond the first two nonstructural genes. Its exclusive presence in pneumovirus among all paramyxovirus suggests a unique function within this small genus. With the aim of understanding its biochemical properties, we investigated this α-helical tetramer by making use of a biophysical approach. We found that the tetramer hydrodynamic radius is considerably extended at high ionic strengths and determined its zinc content to be one atom per monomer. Dissociation-unfolding experiments show a fully reversible and concentration-dependent cooperative transition, but secondary and tertiary structural changes are uncoupled at lower protein concentrations. We detect the presence of a monomeric intermediate, which can be classified as a "late molten globule" with substantial secondary and tertiary structure. Global fittings of experiments from three different probes at two M(2-1) concentrations provide a free energy of dissociation-unfolding of -36.8 ± 0.1 kcal mol(-1), corresponding to a tight dissociation constant of 10(-28) M(3) at pH 7.0. The tetramer affinity is strongly governed by pH, with a free energy change of 13 kcal mol(-1) when pH decreases from 7.0 to 5.0 (K(D) = 10(-18) M(3)). The drastic changes that take place within a pH range compatible with a cellular environment strongly suggest a regulatory effect of pH on M(2-1) structure and biochemical properties, likely affecting transcription and interaction with proteins and RNA.

Kinetic recognition of the retinoblastoma tumor suppressor by a specific protein target.

The retinoblastoma tumor suppressor (Rb) plays a key role in cell cycle control and is linked to various types of human cancer. Rb binds to the LxCxE motif, present in a number of cellular and viral proteins such as AdE1A, SV40 large T-antigen and human papillomavirus (HPV) E7, all instrumental in revealing fundamental mechanisms of tumor suppression, cell cycle control and gene expression. A detailed kinetic study of RbAB binding to the HPV E7 oncoprotein shows that an LxCxE-containing E7 fragment binds through a fast two-state reaction strongly favored by electrostatic interactions. Conversely, full-length E7 binds through a multistep process involving a pre-equilibrium between E7 conformers, a fast electrostatically driven association step guided by the LxCxE motif and a slow conformational rearrangement. This kinetic complexity arises from the conformational plasticity and intrinsically disordered nature of E7 and from multiple interaction surfaces present in both proteins. Affinity differences between E7N domains from high- and low-risk types are explained by their dissociation rates. In fact, since Rb is at the center of a large protein interaction network, fast and tight recognition provides an advantage for disruption by the viral proteins, where the balance of physiological and pathological interactions is dictated by kinetic ligand competition. The localization of the LxCxE motif within an intrinsically disordered domain provides the fast, diffusion-controlled interaction that allows viral proteins to outcompete physiological targets. We describe the interaction mechanism of Rb with a protein ligand, at the same time an LxCxE-containing model target, and a paradigmatic intrinsically disordered viral oncoprotein.