Ana Luísa M. de Carvalho

Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens’ cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

We report the three-dimensional crystal structure of acidic seminal fluid protein (aSFP), a 12.9 kDa poly-peptide of the spermadhesin family isolated from bovine seminal plasma, solved by the multiple isomorphous replacement method and refined with data to 1.9 Angstrom resolution with a final R-factor of 17.3%. aSFP is built by a single CUB domain architecture, a 100 to 110 amino-acid-residue extracellular module found in 16 functionally diverse proteins. The structure of aSFP reveals that the CUB domain displays a beta-sandwich topology organised into two 5-stranded beta-sheets, each of which contain two parallel and four antiparallel strands. The structure of aSFP is almost identical to that of porcine spermadhesins PSP-I and PSP-II, which in turn show limited structural similarity with jellyroll topologies of certain virus capsid proteins. Essentially, topologically conserved residues in these proteins are those internal amino acids forming the hydrophobic core of the CUB and the jellyroll domains, suggesting their importance in maintaining the integrity of these protein folds, On the other hand, the structure of aSFP shows structural features that are unique to this protein and which may provide a structural ground for understanding the distinct biological properties of different members of the spermadhesin protein family. (C) 1997 Academic Press Limited.

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 Å. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

Spermadhesins, 12,000-14,000 M-r mammalian proteins, include lectins involved in sperm-egg binding and display a single CUB domain architecture. We report the crystal structures of porcine seminal plasma PSP-I/PSP-II, a heterodimer of two glycosylated spermadhesins. and bovine aSFP at 2.4 Angstrom and 1.9 Angstrom resolution respectively.

{Bovine acidic seminal fluid protein (aSFP) is a 12.9 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm(3) diffract to beyond 1.9 Angstrom resolution and belong to space group P2(1)2(1)2, with cell parameters a = 52.4 Angstrom

During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin–dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin–dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.

Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.

Cellulosomes are highly efficient nanomachines that play a fundamental role during the anaerobic deconstruction of complex plant cell wall carbohydrates. The assembly of these complex nanomachines results from the very tight binding of repetitive cohesin modules, located in a noncatalytic molecular scaffold, and dockerin domains located at the C-terminus of the enzyme components of the cellulosome. The number of enzymes found in a cellulosome varies but may reach more than 100 catalytic subunits if cellulosomes are further organized in polycellulosomes, through a second type of cohesin-dockerin interaction. Structural studies have revealed how the cohesin-dockerin interaction mediates cellulosome assembly and cell-surface attachment, while retaining the flexibility required to potentiate catalytic synergy within the complex. Methods that might be applied for the production, purification, and structure determination of cohesin-dockerin complexes are described here. Copyright 2012 Elsevier Inc. All rights reserved.

Cellulosomes are highly elaborate multi-enzyme complexes of Carbohydrate Active enZYmes (CAZYmes) secreted by cellulolytic microorganisms, which very effectively degrade the most abundant polymers on Earth, cellulose and hemicelluloses. Cellulosome assembly requires that a non-catalytic dockerin module found in cellulosomal enzymes binds to one of the various cohesin domains located in a large molecular scaffold called Scaffoldin. A diversity of cohesin -dockerin binding specificities have been described, the combination of which may result in complex plant cell wall degrading systems, maximising the synergy between enzymes in order to improve catalytic efficiency. Structural studies have allowed the spatial flexibility inherent to the cellulosomal system to be determined. Recent progress achieved from the study of the fundamental cohesin and dockerin units involved in cellulosome assembly will be reviewed.

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

We show that a physical trigger, a non-ionizing infrared (IR) radiation at wavelengths strongly absorbed by liquid water, can be used to induce and kinetically control protein (periodic) self-assembly in solution. This phenomenon is explained by considering the effect of IR light on the structuring of protein interfacial water. Our results indicate that the IR radiation can promote enhanced mutual correlations of water molecules in the protein hydration shell. We report on the radiation-induced increase in both the strength and cooperativeness of H-bonds. The presence of a structured dipolar hydration layer can lead to attractive interactions between like-charged biomacromolecules in solution (and crystal nucleation events). Furthermore, our study suggests that enveloping the protein within a layer of structured solvent (an effect enhanced by IR light) can prevent the protein non-specific aggregation favoring periodic self-assembly. Recognizing the ability to affect protein-water interactions by means of IR radiation may have important implications for biological and bio-inspired systems.

Nucleation is a critical step determining the outcome of the entire crystallization process. Finding an effective nucleant for protein crystallization is of utmost importance for structural biology. The latter relies on good-quality crystals to solve the three-dimensional structures of macromolecules. In this study we show that crystalline barium sulfate (BaSO4) with an etched and/or ionic liquid (IL)-functionalized surface (1) can induce protein nucleation at concentrations well below the concentration needed to promote crystal growth under control conditions, (2) can shorten the nucleation time, (3) can increase the growth rate, and finally (4) may help to improve the protein crystal morphology. These effects were shown for lysozyme, RNase A, trypsin, proteinase K, myoglobin, and hemoglobin. Therefore, the use of BaSO4 particles enables us to reduce the amount of protein in crystallization trials and increases the chance of obtaining protein crystals of the desired quality. In the context of the underlying mechanism, it is shown that the protein-solid contact formation is governed by the interaction of the polar compartments of the biomacromolecule with the support. The tendency of a protein to concentrate near the solid surface is enhanced by both the hydrophobicity of the protein and that of the surface (tuned by the functionalizing IL). These mechanisms of interaction of biomacromolecules with inorganic hydrophilic solids correspond to the principles of amphiphilic IL-mineral interactions.

After more than one hundred and thirty thousand protein structures determined by X-ray crystallography{,} the challenge of protein crystallization for 3D structure determination remains. In the quest for additives for efficient protein crystallization{,} inorganic materials emerge as an alternative. Magnetic particles (MPs) are versatile inorganic materials{,} easy to use{,} modify and manipulate in a wide range of biological assays. The potential of using functionalised MPs as crystallization chaperones for protein crystallization was shown in this work. MPs with distinct coatings were rationally designed to promote protein crystallization by affinity-triggered heterogeneous nucleation. Hen egg white lysozyme (HEWL) and trypsin{,} were crystallized in the presence of MPs either bare or coated with a polysaccharide (chitin) or a protein (casein){,} respectively. The addition of MPs was characterized in terms of bound protein to the MPs{,} crystal morphology{,} time-lapse of crystal emergence{,} crystallization yield fold change and crystal diffraction quality for structure determination. The MPs additives have shown to bind to the respective target protein{,} and to promote nucleation and crystal growth without compromising crystal morphology. On the other hand{,} MPs addition led to faster detectable crystal emergence and up to 13 times higher crystallization yield{,} addressing some the challenges in protein crystallization{,} the main bottleneck of macromolecular crystallography. Structure determination of the protein crystallized in the presence of MPs revealed that the structural characteristics of the protein remained unchanged{,} as shown by the superposition with PDB annotated proteins. Moreover{,} and unlike most reported cases{,} it was possible to exclude the inhibitor benzamidine during trypsin crystallisation{,} which is a remarkable result opening new prospects in enzyme engineering and drug design. Our results show that MPs coated with affinity ligands to target proteins can be used as controlled and tailor-made crystallization inducers.