Structural characterization of PaaX, the main repressor of the phenylacetate degradation pathway in Escherichia coli W: A novel fold of transcription regulator proteins.

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer-dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.

Zinc Differentially Modulates the Assembly of Soluble and Polymerized Vimentin.

The intermediate filament protein vimentin constitutes a critical sensor for electrophilic and oxidative stress. We previously showed that vimentin interacts with zinc, which affects its assembly and redox sensing. Here, we used vimentin wt and C328S, an oxidation-resistant mutant showing improved NaCl-induced polymerization, to assess the impact of zinc on soluble and polymerized vimentin by light scattering and electron microscopy. Zinc acts as a switch, reversibly inducing the formation of vimentin oligomeric species. High zinc concentrations elicit optically-detectable vimentin structures with a characteristic morphology depending on the support. These effects also occur in vimentin C328S, but are not mimicked by magnesium. Treatment of vimentin with micromolar ZnCl2 induces fibril-like particles that do not assemble into filaments, but form aggregates upon subsequent addition of NaCl. In contrast, when added to NaCl-polymerized vimentin, zinc increases the diameter or induces lateral association of vimentin wt filaments. Remarkably, these effects are absent or attenuated in vimentin C328S filaments. Therefore, the zinc-vimentin interaction depends on the chemical environment and on the assembly state of the protein, leading to atypical polymerization of soluble vimentin, likely through electrostatic interactions, or to broadening and lateral association of preformed filaments through mechanisms requiring the cysteine residue. Thus, the impact of zinc on vimentin assembly and redox regulation is envisaged.

Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1.

Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts. Rosellinia necatrix quadrivirus 1 (RnQV1), the type species of the family Quadriviridae, is a dsRNA fungal virus with a multipartite genome consisting of four monocistronic segments (segments 1 to 4). dsRNA-2 and dsRNA-4 encode two CPs (P2 and P4, respectively), which coassemble into ∼450-Å-diameter capsids. We used three-dimensional cryo-electron microscopy combined with complementary biophysical techniques to determine the structures of RnQV1 virion strains W1075 and W1118. RnQV1 has a quadripartite genome, and the capsid is based on a single-shelled T=1 lattice built of P2-P4 dimers. Whereas the RnQV1-W1118 capsid is built of full-length CP, P2 and P4 of RnQV1-W1075 are cleaved into several polypeptides, maintaining the capsid structural organization. RnQV1 heterodimers have a quaternary organization similar to that of homodimers of reoviruses and other dsRNA mycoviruses. The RnQV1 capsid is the first T=1 capsid with a heterodimer as an asymmetric unit reported to date and follows the architectural principle for dsRNA viruses that a 120-subunit capsid is a conserved assembly that supports dsRNA replication and organization. IMPORTANCE: Given their importance to health, members of the family Reoviridae are the basis of most structural and functional studies and provide much of our knowledge of dsRNA viruses. Analysis of bacterial, protozoal, and fungal dsRNA viruses has improved our understanding of their structure, function, and evolution, as well. Here, we studied a dsRNA virus that infects the fungus Rosellinia necatrix, an ascomycete that is pathogenic to a wide range of plants. Using three-dimensional cryo-electron microscopy and analytical ultracentrifugation analysis, we determined the structure and stoichiometry of Rosellinia necatrix quadrivirus 1 (RnQV1). The RnQV1 capsid is a T=1 capsid with 60 heterodimers as the asymmetric units. The large amount of genetic information used by RnQV1 to construct a simple T=1 capsid is probably related to the numerous virus-host and virus-virus interactions that it must face in its life cycle, which lacks an extracellular phase.

Crowding Modulates the Conformation, Affinity, and Activity of the Components of the Bacterial Disaggregase Machinery.

Chaperone-mediated protein aggregate reactivation is a complex reaction that depends on the sequential association of molecular chaperones on their interaction with protein aggregates and on substrate refolding. This process could be modulated by the highly crowded intracellular environment, which is known to affect protein conformational change, enzymatic activity, and protein-protein interactions. Here, we report that molecular crowding shapes the chaperone activity of bacterial disaggregase composed of the DnaK system (DnaK, DnaJ, and GrpE) and the molecular motor ClpB. A combination of biophysical and biochemical methods shows that the excluded volume conditions modify the conformation of DnaK and DnaJ without affecting that of GrpE. These crowding-induced conformational rearrangements activate DnaK, enhance the affinity of DnaK for DnaJ, but not for GrpE, and increase the sensitivity of the chaperone activity to cochaperone concentration, explaining the tight control of their relative intracellular amounts. Furthermore, crowding-mediated disordering of the G/F domain of DnaJ facilitates the reversible formation of intermolecular DnaJ conglomerates. These assemblies could drive the formation of Hsp70 clusters at the aggregate surface with the consequent enhancement of the disaggregation efficiency through their coordinated action via entropic pulling. Finally, crowding helps ClpB to outcompete GrpE for DnaK binding, a key aspect of DnaK/ClpB cooperation given the low affinity of the disaggregase for DnaK. Excluded volume conditions promote the formation of the bichaperone complex that disentangles aggregates, enhancing the efficiency of the disaggregation reaction.

RepA-WH1, the agent of an amyloid proteinopathy in bacteria, builds oligomeric pores through lipid vesicles.

RepA-WH1 is a disease-unrelated protein that recapitulates in bacteria key aspects of human amyloid proteinopathies: i) It undergoes ligand-promoted amyloidogenesis in vitro; ii) its aggregates are able to seed/template amyloidosis on soluble protein molecules; iii) its conformation is modulated by Hsp70 chaperones in vivo, generating transmissible amyloid strains; and iv) causes proliferative senescence. Membrane disruption by amyloidogenic oligomers has been found for most proteins causing human neurodegenerative diseases. Here we report that, as for PrP prion and α-synuclein, acidic phospholipids also promote RepA-WH1 amyloidogenesis in vitro. RepA-WH1 molecules bind to liposomes, where the protein assembles oligomeric membrane pores. Fluorescent tracer molecules entrapped in the lumen of the vesicles leak through these pores and RepA-WH1 can then form large aggregates on the surface of the vesicles without inducing their lysis. These findings prove that it is feasible to generate in vitro a synthetic proteinopathy with a minimal set of cytomimetic components and support the view that cell membranes are primary targets in protein amyloidoses.

Sedimentation Equilibrium Analysis of ClpB Self-Association in Diluted and Crowded Solutions.

ClpB belongs to the Hsp100 family of ring-forming heat-shock proteins involved in degradation of unfolded/misfolded proteins and in reactivation of protein aggregates. ClpB monomers reversibly associate to form the hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. Here, we summarize the use of sedimentation equilibrium approaches, complemented with sedimentation velocity and composition-gradient static light scattering measurements, to study the self-association properties of ClpB in dilute and crowded solutions. As the functional unit of ClpB is the hexamer, we study the effect of environmental factors, i.e., ionic strength and natural ligands, in the association equilibrium of ClpB as well as the role of the flexible N-terminal and M domains of the protein in the self-association process. The application of the nonideal sedimentation equilibrium technique to measure the effects of volume exclusion, reproducing in part the natural crowded conditions inside a cell, on the self-association and on the stability of the oligomeric species of the disaggregase will be described. Finally, the biochemical and physiological implications of these studies and future experimental challenges to eventually reconstitute minimal disaggregating machineries will be discussed.

Small Molecule Inhibition of ERK Dimerization Prevents Tumorigenesis by RAS-ERK Pathway Oncogenes.

Nearly 50% of human malignancies exhibit unregulated RAS-ERK signaling; inhibiting it is a valid strategy for antineoplastic intervention. Upon activation, ERK dimerize, which is essential for ERK extranuclear, but not for nuclear, signaling. Here, we describe a small molecule inhibitor for ERK dimerization that, without affecting ERK phosphorylation, forestalls tumorigenesis driven by RAS-ERK pathway oncogenes. This compound is unaffected by resistance mechanisms that hamper classical RAS-ERK pathway inhibitors. Thus, ERK dimerization inhibitors provide the proof of principle for two understudied concepts in cancer therapy: (1) the blockade of sub-localization-specific sub-signals, rather than total signals, as a means of impeding oncogenic RAS-ERK signaling and (2) targeting regulatory protein-protein interactions, rather than catalytic activities, as an approach for producing effective antitumor agents.

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits.

The effects of Kil peptide from bacteriophage λ on the assembly of Escherichia coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has recently been identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here, we show that this antagonist blocks FtsZ assembly into GTP-dependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in guanosine-5'-[(α,ß)-methyleno]triphosphate are considerably less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection.

Role of the FtsA C terminus as a switch for polymerization and membrane association.

UNLABELLED: Together with ATP, the C-terminal region of the essential streptococcal FtsA protein acts as an intramolecular switch to promote its polymerization and attachment to the membrane. During septation, FtsA is known to anchor the constricting FtsZ ring and, subsequently, the divisome to the membrane. Truncation of the C terminus of the streptococcal FtsA (FtsAΔCt) facilitates a more rapid ATP-dependent polymerization in solution than is seen with the full-length protein (FtsA(+)). The FtsAΔCt polymers are more organized and compact than those formed in solution by FtsA(+), resembling the shape of the membrane-associated FtsA(+) polymers. We find that ATP, besides being needed for polymerization, is required for the attachment of FtsA(+) to lipid monolayers and to vesicle membranes. We propose a model in which the binding of ATP activates a switch favoring the polymerization of FtsA and at the same time driving the amphipathic helix at its C terminus to become attached to the membrane. Conversely, when FtsA is in the cytoplasm, the C terminus is not engaged in the attachment to the membrane, and it obstructs polymerization. ATP-dependent polymerization of FtsA inside membrane vesicles causes vesicle shrinkage, suggesting that, besides providing a membrane attachment for FtsZ, the FtsA C terminus may also introduce local alterations in the membrane to facilitate septation. IMPORTANCE: FtsA is a protein needed in many bacteria to construct a septum that divides one fully grown cell, producing two daughters. We show that the region located at the C-terminal end of the Streptococcus pneumoniae FtsA protein works as a switch triggered by ATP, a molecule that stores energy. This region contains an amphipathic helix that obstructs the assembly of FtsA into polymers in the cytoplasm. In the presence of ATP, the obstruction is removed by switching the position of the helix. The switch directs the helix to the membrane and simultaneously facilitates the polymerization of the protein. The accumulation of FtsA molecules at the membrane causes distortions, an effect produced also by proteins such as MinD, MreB, and SepF that also contain amphipathic helixes as membrane attachment devices. In the case of FtsA, these distortions may also facilitate the initial events that lead to the division of bacteria.

Crowding activates ClpB and enhances its association with DnaK for efficient protein aggregate reactivation.

Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner.

A 17-residue sequence from the matrix metalloproteinase-9 (MMP-9) hemopexin domain binds α4ß1 integrin and inhibits MMP-9-induced functions in chronic lymphocytic leukemia B cells.

We previously showed that pro-matrix metalloproteinase-9 (proMMP-9) binds to B chronic lymphocytic leukemia (B-CLL) cells and contributes to B-CLL progression by regulating cell migration and survival. Induction of cell survival involves a non-proteolytic mechanism and the proMMP-9 hemopexin domain (PEX9). To help design specific inhibitors of proMMP-9-cell binding, we have now characterized B-CLL cell interaction with the isolated PEX9. B-CLL cells bound soluble and immobilized GST-PEX9, but not GST, and binding was mediated by α4ß1 integrin. The ability to recognize PEX9 was observed in all 20 primary samples studied irrespective of their clinical stage or prognostic marker phenotype. By preparing truncated forms of GST-PEX9 containing structural blades B1B2 or B3B4, we have identified B3B4 as the primary α4ß1 integrin-interacting region within PEX9. Overlapping synthetic peptides spanning B3B4 were then tested in functional assays. Peptide P3 (FPGVPLDTHDVFQYREKAYFC), a sequence present in B4 or smaller versions of this sequence (peptides P3a/P3b), inhibited B-CLL cell adhesion to GST-PEX9 or proMMP-9, with IC(50) values of 138 and 279 µm, respectively. Mutating the two aspartate residues to alanine rendered the peptides inactive. An anti-P3 antibody also inhibited adhesion to GST-PEX9 and proMMP-9. GST-PEX9, GST-B3B4, and P3/P3a/P3b peptides inhibited B-CLL cell transendothelial migration, whereas the mutated peptide did not. B-CLL cell incubation with GST-PEX9 induced intracellular survival signals, namely Lyn phosphorylation and Mcl-1 up-regulation, and this was also prevented by the P3 peptides. The P3 sequence may, therefore, constitute an excellent target to prevent proMMP-9 contribution to B-CLL pathogenesis.

Key role of two terminal domains in the bidirectional polymerization of FtsA protein.

The effect of two different truncations involving either the 1C domain or the simultaneous absence of the S12-13 ß-strands of the FtsA protein from Streptococcus pneumoniae, located at opposite terminal sides in the molecular structure, suggests that they are essential for ATP-dependent polymerization. These two truncated proteins are not able to polymerize themselves but can be incorporated to some extent into the FtsA(+) polymers during the assembling process. Consequently, they block the growth of the FtsA(+) polymers and slow down the polymerization rate. The combined action of the two truncated proteins produces an additive effect on the inhibition of FtsA(+) polymerization, indicating that each truncation affects a different interaction site within the FtsA molecule.

Effects of macromolecular crowding on the inhibition of virus assembly and virus-cell receptor recognition.

Biological fluids contain a very high total concentration of macromolecules that leads to volume exclusion by one molecule to another. Theory and experiment have shown that this condition, termed macromolecular crowding, can have significant effects on molecular recognition. However, the influence of molecular crowding on recognition events involving virus particles, and their inhibition by antiviral compounds, is virtually unexplored. Among these processes, capsid self-assembly during viral morphogenesis and capsid-cell receptor recognition during virus entry into cells are receiving increasing attention as targets for the development of new antiviral drugs. In this study, we have analyzed the effect of macromolecular crowding on the inhibition of these two processes by peptides. Macromolecular crowding led to a significant reduction in the inhibitory activity of: 1), a capsid-binding peptide and a small capsid protein domain that interfere with assembly of the human immunodeficiency virus capsid, and 2), a RGD-containing peptide able to block the interaction between foot-and-mouth disease virus and receptor molecules on the host cell membrane (in this case, the effect was dependent on the conditions used). The results, discussed in the light of macromolecular crowding theory, are relevant for a quantitative understanding of molecular recognition processes during virus infection and its inhibition.

Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: a bona fide tracer.

Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF-Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group's side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF-Cys2-heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF-Cys2-heparin complex might represent a biologically relevant complex in physiological media.

The conjugative DNA translocase TrwB is a structure-specific DNA-binding protein.

TrwB is a DNA-dependent ATPase involved in DNA transport during bacterial conjugation. The protein presents structural similarity to hexameric molecular motors such as F(1)-ATPase, FtsK, or ring helicases, suggesting that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump single-stranded DNA through its central channel. In this work, we have carried out an extensive analysis with various DNA substrates to determine the preferred substrate for TrwB. Oligonucleotides with G-rich sequences forming G4 DNA structures were the optimal substrates for TrwB ATPase activity. The protein bound with 100-fold higher affinity to G4 DNA than to single-stranded DNA of the same sequence. Moreover, TrwB formed oligomeric protein complexes only with oligonucleotides presenting such a G-quadruplex DNA structure, consistent with stoichiometry of six TrwB monomers to G4 DNA, as demonstrated by gel filtration chromatography and analytical ultracentrifugation experiments. A protein-DNA complex was also formed with unstructured oligonucleotides, but the molecular mass corresponded to one monomer protein bound to one oligonucleotide molecule. Sequences capable of forming G-quadruplex structures are widespread through genomes and are thought to play a biological function in transcriptional regulation. They form stable structures that can obstruct DNA replication, requiring the action of specific helicases to resolve them. Nevertheless, TrwB displayed no G4 DNA unwinding activity. These observations are discussed in terms of a possible role for TrwB in recognizing G-quadruplex structures as loading sites on the DNA.

Infectious bursal disease virus is an icosahedral polyploid dsRNA virus.

Viruses are a paradigm of the economy of genome resources, reflected in their multiplication strategy and for their own structure. Although there is enormous structural diversity, the viral genome is always enclosed within a proteinaceous coat, and most virus species are haploid; the only exception to this rule are the highly pleomorphic enveloped viruses. We performed an in-depth characterization of infectious bursal disease virus (IBDV), a non-enveloped icosahedral dsRNA virus with a bisegmented genome. Up to 6 natural populations can be purified, which share a similar protein composition but show higher sedimentation coefficients as particle density increases. Stoichiometry analysis of their genome indicated that these biophysical differences correlate with the copy number of dsRNA segments inside the viral capsid. This is a demonstration of a functional polyploid icosahedral dsRNA virus. We show that IBDV particles with greater genome copy number have higher infectivity rates. Our results show an unprecedented replicative strategy for dsRNA viruses and suggest that birnaviruses are living viral entities encompassing numerous functional and structural characteristics of positive and negative ssRNA viruses.

ATPase activity and oligomeric state of TrwK, the VirB4 homologue of the plasmid R388 type IV secretion system.

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein.

Structural and functional differences among human surfactant proteins SP-A1, SP-A2 and co-expressed SP-A1/SP-A2: role of supratrimeric oligomerization.

SP-A (surfactant protein A) is a membrane-associated SP that helps to maintain the lung in a sterile and non-inflamed state. Unlike SP-As from other mammalian species, human SP-A consists of two functional gene products: SP-A1 and SP-A2. In all the functions examined, recombinant human SP-A1 invariably exhibits lower biological activity than SP-A2. The objective of the present study was to investigate why SP-A2 possesses greater biological activity than SP-A1 and what advantage accrues to having two polypeptide chains instead of one. We analysed structural and functional characteristics of recombinant baculovirus-derived SP-A1, SP-A2 and co-expressed SP-A1/SP-A2 using a wide array of experimental approaches such as analytical ultracentrifugation, DSC (differential scanning calorimetry) and fluorescence. We found that the extent of supratrimeric assembly is much lower in SP-A1 than SP-A2. However, the resistance to proteolysis is greater for SP-A1 than for SP-A2. Co-expressed SP-A1/SP-A2 had greater thermal stability than SP-A1 and SP-A2 and exhibited properties of each protein. On the one hand, SP-A1/SP-A2, like SP-A2, had a higher degree of oligomerization than SP-A1, and consequently had lower K(d) for binding to bacterial Re-LPS (rough lipopolysaccharide), higher self-association in the presence of calcium and greater capability to aggregate Re-LPS and phospholipids than SP-A1. On the other hand, SP-A1/SP-A2, like SP-A1, was more resistant to trypsin degradation than SP-A2. Finally, the importance of the supratrimeric assembly for SP-A immunomodulatory function is discussed.

Structural and functional analysis of phi29 p16.7C dimerization mutants: identification of a novel aromatic cage dimerization motif.

Prokaryotic DNA replication is compartmentalized at the cellular membrane. The Bacillus subtilis phage varphi29-encoded membrane protein p16.7 is one of the few proteins known to be involved in the organization of prokaryotic membrane-associated DNA replication. The functional DNA binding domain of p16.7 is constituted by its C-terminal half, p16.7C, which forms high affinity dimers in solution and which can form higher order oligomers. Recently, the solution and crystal structures of p16.7C and the crystal structure of the p16.7C-DNA complex have been solved. Here, we have studied the p16.7C dimerization process and the structural and functional roles of p16.7 residues Trp-116 and Asn-120 and its last nine C-terminal amino acids, which form an extended tail. The results obtained show that transition of folded dimers into unfolded monomers occurs without stable intermediates and that both Trp-116 and the C-terminal tail are important for dimerization and functionality of p16.7C. Residue Trp-116 is involved in formation of a novel aromatic cage dimerization motif, which we call "Pro cage." Finally, whereas residue Asn-120 plays a minor role in p16.7C dimerization, we show that it is critical for both oligomerization and DNA binding, providing further evidence that DNA binding and oligomerization of p16.7C are coupled processes.