Unveiling CD59-Antibody Interactions to Design Paratope-Mimicking Peptides for Complement Modulation.

CD59 is an abundant immuno-regulatory human protein that protects cells from damage by inhibiting the complement system. CD59 inhibits the assembly of the Membrane Attack Complex (MAC), the bactericidal pore-forming toxin of the innate immune system. In addition, several pathogenic viruses, including HIV-1, escape complement-mediated virolysis by incorporating this complement inhibitor in their own viral envelope. This makes human pathogenic viruses, such as HIV-1, not neutralised by the complement in human fluids. CD59 is also overexpressed in several cancer cells to resist the complement attack. Consistent with its importance as a therapeutical target, CD59-targeting antibodies have been proven to be successful in hindering HIV-1 growth and counteracting the effect of complement inhibition by specific cancer cells. In this work, we make use of bioinformatics and computational tools to identify CD59 interactions with blocking antibodies and to describe molecular details of the paratope-epitope interface. Based on this information, we design and produce paratope-mimicking bicyclic peptides able to target CD59. Our results set the basis for the development of antibody-mimicking small molecules targeting CD59 with potential therapeutic interest as complement activators.

Structure-Based Development of SARS-CoV-2 Spike Interactors.

Coronaviruses, including SARS-CoV-2 (the etiological agent of the current COVID-19 pandemic), rely on the surface spike glycoprotein to access the host cells, mainly through the interaction of their receptor-binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2). Therefore, molecular entities able to interfere with the binding of the SARS-CoV-2 spike protein to ACE2 have great potential to inhibit viral entry. Starting from the available structural data on the interaction between SARS-CoV-2 spike protein and the host ACE2 receptor, we engineered a set of soluble and stable spike interactors, here denoted as S-plugs. Starting from the prototype S-plug, we adopted a computational approach by combining stability prediction, associated to single-point mutations, with molecular dynamics to enhance both S-plug thermostability and binding affinity to the spike protein. The best developed molecule, S-plug3, possesses a highly stable α-helical con-formation (with melting temperature Tm of 54 °C) and can interact with the spike RBD and S1 domains with similar low nanomolar affinities. Importantly, S-plug3 exposes the spike RBD to almost the same interface as the human ACE2 receptor, aimed at the recognition of all ACE2-accessing coronaviruses. Consistently, S-plug3 preserves a low nanomolar dissociation constant with the delta B.1.617.2 variant of SARS-CoV-2 spike protein (KD = 29.2 ± 0.6 nM). Taken together, we provide valid starting data for the development of therapeutical and diagnostic tools against coronaviruses accessing through ACE2.

Members of the GADD45 Protein Family Show Distinct Propensities to form Toxic Amyloid-Like Aggregates in Physiological Conditions.

The three members (GADD45α, GADD45ß, and GADD45γ) of the growth arrest and DNA damage-inducible 45 (GADD45) protein family are involved in a myriad of diversified cellular functions. With the aim of unravelling analogies and differences, we performed comparative biochemical and biophysical analyses on the three proteins. The characterization and quantification of their binding to the MKK7 kinase, a validated functional partner of GADD45ß, indicate that GADD45α and GADD45γ are strong interactors of the kinase. Despite their remarkable sequence similarity, the three proteins present rather distinct biophysical properties. Indeed, while GADD45ß and GADD45γ are marginally stable at physiological temperatures, GADD45α presents the Tm value expected for a protein isolated from a mesophilic organism. Surprisingly, GADD45α and GADD45ß, when heated, form high-molecular weight species that exhibit features (ThT binding and intrinsic label-free UV/visible fluorescence) proper of amyloid-like aggregates. Cell viability studies demonstrate that they are endowed with a remarkable toxicity against SHSY-5Y and HepG2 cells. The very uncommon property of GADD45ß to form cytotoxic species in near-physiological conditions represents a puzzling finding with potential functional implications. Finally, the low stability and/or the propensity to form toxic species of GADD45 proteins constitute important features that should be considered in interpreting their many functions.

On the extraordinary pressure stability of the Thermotoga maritima arginine binding protein and its folded fragments - a high-pressure FTIR spectroscopy study.

The arginine binding protein from T. maritima (ArgBP) exhibits several distinctive biophysical and structural properties. Here we show that ArgBP is also endowed with a ramarkable pressure stability as it undergoes minor structural changes only, even at 10 kbar. A similar stability is also observed for its folded fragments (truncated monomer and individual domains). A survey of literature data on the pressure stability of proteins highlights the uncommon behavior of ArgBP.

Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima.

Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors.

Collagen degradation in tuberculosis pathogenesis: the biochemical consequences of hosting an undesired guest.

The scenario of chemical reactions prompted by the infection by Mycobacterium tuberculosis is huge. The infection generates a localized inflammatory response, with the recruitment of neutrophils, monocytes, and T-lymphocytes. Consequences of this immune reaction can be the eradication or containment of the infection, but these events can be deleterious to the host inasmuch as lung tissue can be destroyed. Indeed, a hallmark of tuberculosis (TB) is the formation of lung cavities, which increase disease development and transmission, as they are sites of high mycobacterial burden. Pulmonary cavitation is associated with antibiotic failure and the emergence of antibiotic resistance. For cavities to form, M. tuberculosis induces the overexpression of host proteases, like matrix metalloproteinases and cathepsin, which are secreted from monocyte-derived cells, neutrophils, and stromal cells. These proteases destroy the lung parenchyma, in particular the collagen constituent of the extracellular matrix (ECM). Namely, in an attempt to destroy infected cells, the immune reactions prompted by mycobacterial infections induce the destruction of vital regions of the lung, in a process that can become fatal. Here, we review structure and function of the main molecular actors of ECM degradation due to M. tuberculosis infection and the proposed mechanisms of tissue destruction, mainly attacking fibrillar collagen. Importantly, enzymes responsible for collagen destruction are emerging as key targets for adjunctive therapies to limit immunopathology in TB.

Domain swapping dissection in Thermotoga maritima arginine binding protein: How structural flexibility may compensate destabilization.

Thermotoga maritima Arginine Binding Protein (TmArgBP) is a valuable candidate for arginine biosensing in diagnostics. This protein is endowed with unusual structural properties that include an extraordinary thermal/chemical stability, a domain swapped structure that undergoes large tertiary and quaternary structural transition, and the ability to form non-canonical oligomeric species. As the intrinsic stability of TmArgBP allows for extensive protein manipulations, we here dissected its structure in two parts: its main body deprived of the swapping fragment (TmArgBP20-233) and the C-terminal peptide corresponding to the helical swapping element. Both elements have been characterized independently or in combination using a repertoire of biophysical/structural techniques. Present investigations clearly indicate that TmArgBP20-233 represents a better scaffold for arginine sensing compared to the wild-type protein. Moreover, our data demonstrate that the ligand-free and the ligand-bound forms respond very differently to this helix deletion. This drastic perturbation has an important impact on the ligand-bound form of TmArgBP20-233 stability whereas it barely affects its ligand-free state. The crystallographic structures of these forms provide a rationale to this puzzling observation. Indeed, the arginine-bound state is very rigid and virtually unchanged upon protein truncation. On the other hand, the flexible ligand-free TmArgBP20-233 is able to adopt a novel state as a consequence of the helix deletion. Therefore, the flexibility of the ligand-free form endows this state with a remarkable robustness upon severe perturbations. In this scenario, TmArgBP dissection highlights an intriguing connection between destabilizing/stabilizing effects and the overall flexibility that could operate also in other proteins.

Chemistry of Peptidoglycan in Mycobacterium tuberculosis Life Cycle: An off-the-wall Balance of Synthesis and Degradation.

The cell wall envelope of mycobacteria is structurally distinct from that of both Gram-positive and Gram-negative bacteria. In Mycobacterium tuberculosis, this cell wall has unique structural features and plays a crucial role in drug resistance and macrophage survival under stress conditions. Peptidoglycan is the major constituent of this cell wall, with an important structural role, giving structural strength, and counteracting the osmotic pressure of the cytoplasm. Synthesis of this complex polymer takes place in three stages that occur at three different locations in the cell, from the cytoplasm to the external side of the cell membrane, where polymerization occurs. A fine balance of peptidoglycan synthesis and degradation is responsible for a plethora of molecular mechanisms which are key to the pathogenicity of M. tuberculosis. Enlargement of mycobacterial cells can occur through the synthesis of new peptidoglycan, autolysis of old peptidoglycan, or a combination of both processes. Here, we discuss the chemical aspects of peptidoglycan synthesis and degradation, in relation to metabolic stages of M. tuberculosis. Going from inside the mycobacterial cytoplasm to outside its membrane, we describe the assembly line of peptidoglycan synthesis and polymerization, and continue with its depolymerization events and their consequences on mycobacterial life and resuscitation from dormancy.

The Burkholderia cenocepacia peptidoglycan-associated lipoprotein is involved in epithelial cell attachment and elicitation of inflammation.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic pathogens causing infections in people with cystic fibrosis (CF). Bcc is highly antibiotic resistant, making conventional antibiotic treatment problematic. The identification of novel targets for anti-virulence therapies should improve therapeutic options for infected CF patients. We previously identified that the peptidoglycan-associated lipoprotein (Pal) was immunogenic in Bcc infected CF patients; however, its role in Bcc pathogenesis is unknown. The virulence of a pal deletion mutant (Δpal) in Galleria mellonella was 88-fold reduced (p < .001) compared to wild type. The lipopolysaccharide profiles of wild type and Δpal were identical, indicating no involvement of Pal in O-antigen transport. However, Δpal was more susceptible to polymyxin B. Structural elucidation by X-ray crystallography and calorimetry demonstrated that Pal binds peptidoglycan fragments. Δpal showed a 1.5-fold reduced stimulation of IL-8 in CF epithelial cells relative to wild type (p < .001), demonstrating that Pal is a significant driver of inflammation. The Δpal mutant had reduced binding to CFBE41o- cells, but adhesion of Pal-expressing recombinant E. coli to CFBE41o- cells was enhanced compared to wild-type E. coli (p < .0001), confirming that Pal plays a direct role in host cell attachment. Overall, Bcc Pal mediates host cell attachment and stimulation of cytokine secretion, contributing to Bcc pathogenesis.

The structure of Resuscitation promoting factor B from M. tuberculosis reveals unexpected ubiquitin-like domains.

BACKGROUND: RpfB is a key factor in resuscitation from dormancy of Mycobacterium tuberculosis. This protein is a cell-wall glycosidase, which cleaves cell-wall peptidoglycan. RpfB is structurally complex and is composed of three types of domains, including a catalytic, a G5 and three DUF348 domains. Structural information is currently limited to a portion of the protein including only the catalytic and G5 domains. To gain insights into the structure and function of all domains we have undertaken structural investigations on a large protein fragment containing all three types of domains that constitute RpfB (RpfB3D). METHODS: The structural features of RpfB3D have been investigated combining x-ray crystallography and biophysical studies. RESULTS AND CONCLUSIONS: The crystal structure of RpfB3D provides the first structural characterization of a DUF348 domain and revealed an unexpected structural relationship with ubiquitin. The crystal structure also provides specific structural features of these domains explaining their frequent association with G5 domains. GENERAL SIGNIFICANCE: Results provided novel insights into the mechanism of peptidoglycan degradation necessary to the resuscitation of M. tuberculosis. Features of the DUF348 domain add structural data to a large set of proteins embedding this domain. Based on its structural similarity to ubiquitin and frequent association to the G5 domain, we propose to name this domain as G5-linked-Ubiquitin-like domain, UBLG5.

Proline 235 plays a key role in the regulation of the oligomeric states of Thermotoga maritima Arginine Binding Protein.

The Arginine Binding Protein isolated from Thermotoga maritima (TmArgBP) is a protein endowed with several peculiar properties. We have previously shown that TmArgBP dimerization is a consequence of the swapping of the C-terminal helix. Here we explored the structural determinants of TmArgBP domain swapping and oligomerization. In particular, we report a mutational analysis of the residue Pro235, which is located in the hinge region of the swapping dimer. This residue was either replaced with a Gly-Lys dipeptide (TmArgBP(P235GK)) or a Gly residue (TmArgBP(P235G)). Different forms of these mutants were generated and extensively characterized using biophysical techniques. For both TmArgBP(P235GK) and TmArgBP(P235G) mutants, the occurrence of multiple oligomerization states (monomers, dimers and trimers) was detected. The formation of well-folded monomeric forms for these mutants indicates that the dimerization through C-terminal domain swapping observed in wild-type TmArgBP is driven by conformational restraints imposed by the presence of Pro235 in the hinge region. Molecular dynamics studies corroborate this observation by showing that Gly235 assumes conformational states forbidden for Pro residues in the TmArgBP(P235G) monomer. Unexpectedly, the trimeric forms present: (a) peculiar circular dichroism spectra, (b) a great susceptibility to heating, and (c) the ability to bind the Thioflavin T dye. The present findings clearly demonstrate that single-point mutations have an important impact on the TmArgBP oligomerization process. In a wider context, they also indicate that proteins endowed with an intrinsic propensity to swap have an easy access to states with altered structural and, possibly, functional properties.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

UNLABELLED: The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a ß-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE: Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a ß-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization.

Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile122 in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)8 induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)8-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNß1,4GlcNAc)4, which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1.

Differential thermodynamic behaviours of the extra-cellular regions of two Ser/Thr PrkC kinases revealed by calorimetric studies.

Eukaryotic-type Ser/Thr protein-kinases are critical mediators of developmental changes and host pathogen interactions in bacteria. Although with lower abundance compared to their homologues from eukaryotes, Ser/Thr protein-kinases (STPK) are widespread in gram positive bacteria, where they regulate several cellular functions. STPKs belong to the protein kinase family named as one-component signal transduction systems, which combine both sensing and regulating properties. Thermodynamic investigations of sensing extra-cellular portions of two important Ser-Thr kinases, PrkC, from Staphylococcus aureus and Bacillus subtilis were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements, coupled with modelling studies. The study of thermodynamic properties of the two domains is challenging since they share a modular domain organization. Consistently, DSC and CD data show that they present similar thermodynamic behaviours and that folding/unfolding transitions do not fit a two-state folding model. However, the thermal unfolding of the two proteins is differentially sensitive to pH. In particular, their unfolding is characteristic of modular structures at the neutral pH, with independent contributions of individual domains to folding. Differently, a cooperative unfolding is evidenced at acidic pH for the B. subtilis member, suggesting that a significant interaction between domains becomes valuable.

Mutational and structural study of RipA, a key enzyme in Mycobacterium tuberculosis cell division: evidence for the L-to-D inversion of configuration of the catalytic cysteine.

RipA is a key cysteine protease of Mycobacterium tuberculosis as it is responsible for bacterial daughter-cell separation. Although it is an important target for antimicrobial development, its mechanism of action and its interaction pattern with its substrate are hitherto unknown. By combining crystallographic and mutational studies with functional assays and molecular modelling, it is shown that the catalytic activity of the enzyme relies on a Cys-His-Glu triad and the impact of the mutation of each residue of the triad on the structure and function of RipA is analysed. Unexpectedly, the crystallographic analyses reveal that mutation of the glutamic acid to alanine results in inversion of the configuration of the catalytic cysteine. The consequent burial of the catalytic cysteine side chain explains the enzyme inactivation upon mutation. These data point to a novel role of the acidic residue often present in the triad of cysteine proteases as a supervisor of cysteine configuration through preservation of the local structural integrity.

Structural and binding properties of the PASTA domain of PonA2, a key penicillin binding protein from Mycobacterium tuberculosis.

PonA2 is one of the two class A penicillin binding proteins of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. It plays a complex role in mycobacterial physiology and is spotted as a promising target for inhibitors. PonA2 is involved in adaptation of M. tuberculosis to dormancy, an ability which has been attributed to the presence in its sequence of a C-terminal PASTA domain. Since PASTA modules are typically considered as ß-lactam antibiotic binding domains, we determined the solution structure of the PASTA domain from PonA2 and analyzed its binding properties versus a plethora of potential binders, including the ß-lactam antibiotics, two typical muropeptide mimics, and polymeric peptidoglycan. We show that, despite a high structural similarity with other PASTA domains, the PASTA domain of PonA2 displays different binding properties, as it is not able to bind muropeptides, or ß-lactams, or polymeric peptidoglycan. These results indicate that the role of PASTA domains cannot be generalized, as their specific binding properties strongly depend on surface residues, which are widely variable.

HtpG-A Major Virulence Factor and a Promising Vaccine Antigen against Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading global cause of death f rom an infectious bacterial agent. Therefore, limiting its epidemic spread is a pressing global health priority. The chaperone-like protein HtpG of M. tuberculosis (Mtb) is a large dimeric and multi-domain protein with a key role in Mtb pathogenesis and promising antigenic properties. This dual role, likely associated with the ability of Heat Shock proteins to act both intra- and extra-cellularly, makes HtpG highly exploitable both for drug and vaccine development. This review aims to gather the latest updates in HtpG structure and biological function, with HtpG operating in conjunction with a large number of chaperone molecules of Mtb. Altogether, these molecules help Mtb recovery after exposure to host-like stress by assisting the whole path of protein folding rescue, from the solubilisation of aggregated proteins to their refolding. Also, we highlight the role of structural biology in the development of safer and more effective subunit antigens. The larger availability of structural information on Mtb antigens and a better understanding of the host immune response to TB infection will aid the acceleration of TB vaccine development.

Carbohydrate recognition by RpfB from Mycobacterium tuberculosis unveiled by crystallographic and molecular dynamics analyses.

Resuscitation of Mtb is crucial to the etiology of Tuberculosis, because latent tuberculosis is estimated to affect one-third of the world population. The resuscitation-promoting factor RpfB is mainly responsible for Mtb resuscitation from dormancy. Given the impact of latent Tuberculosis, RpfB represents an interesting target for tuberculosis drug discovery. However, no molecular models of substrate binding and catalysis are hitherto available for this enzyme. Here, we identified key interactions involved in substrate binding to RpfB by combining x-ray diffraction studies and computational approaches. The crystal structure of RpfB catalytic domain in complex with N,N',N"-triacetyl-chitotriose, as described here, provides the first, to our knowledge, atomic representation of ligand recognition by RpfB and demonstrates that the strongest interactions are established by the N-acetylglucosamine moiety in the central region of the enzyme binding cleft. Molecular dynamics analyses provided information on the dynamic behavior of protein-substrate interactions and on the role played by the solvent in RpfB function. These data combined with sequence conservation analysis suggest that Glu-292 is the sole residue crucial for catalysis, implying that RpfB acts via the formation of an oxocarbenium ion rather than a covalent intermediate. Present data represent a solid base for the design of effective drug inhibitors of RpfB. Moreover, homology models were generated for the catalytic domains of all members of the Mtb Rpf family (RpfA-E). The analysis of these models unveiled analogies and differences among the different members of the Rpf protein family.

Structures of free and inhibited forms of the L,D-transpeptidase LdtMt1 from Mycobacterium tuberculosis.

The modelling of peptidoglycan is responsible for key cellular processes in Mycobacterium tuberculosis such as cell growth, division and resuscitation from dormancy. The structure of M. tuberculosis peptidoglycan is atypical since it contains a majority of 3,3 cross-links synthesized by L,D-transpeptidases that replace the 4,3 cross-links formed by the D,D-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these L,D-transpeptidases and in combination with clavulanic acid are bactericidal against extensively drug-resistant M. tuberculosis. Here, crystal structures of the L,D-transpeptidase LdtMt1 from M. tuberculosis in a ligand-free form and in complex with the carbapenem imipenem are reported. Elucidation of the structural features of LdtMt1 unveils analogies and differences between the two key transpeptidases of M. tuberculosis: LdtMt1 and LdtMt2. In addition, the structure of imipenem-inactivated LdtMt1 provides a detailed structural view of the interactions between a carbapenem drug and LdtMt1. By providing the key interactions in the binding of carbapenem to LdtMt1, this work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antitubercular agents against drug-resistant M. tuberculosis.

Role of mutual interactions in the chemical and thermal stability of nucleophosmin NPM1 domains.

Nucleophosmin (NPM1) is a key factor involved in fundamental biological processes. Mutations involving the NPM1 gene are the most frequent molecular alterations in acute myeloid leukemia. Here we report a biophysical characterization of NPM1 and of its domains in order to gain insights into the role that inter-domain interactions plays in the protein stabilization. Thermal denaturation analyses show that the N-terminal domain is endowed with an exceptional thermal stability, as it does not unfold in the investigated temperature range (20-105°C). This finding is corroborated by chemical denaturation experiments showing that this domain is not significantly affected by the addition of 8M urea. These results are consistent with the chaperone function of NPM1. In line with literature data, the other folded domain of the NPM1, a 3-helix bundle domain located at the C-terminus, shows a lower stability. Interestingly, the chemical and thermal stability of this latter domain, which embeds natural mutations related to acute myeloid leukemia, is influenced by the presence of other regions of the protein. Small but significant stabilizations of the C-terminal 3-helix bundle are provided by the adjacent unfolded fragment as well as by the rest of the protein.