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.

Klebsiella phage KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against K. pneumoniae.

IMPORTANCE: In this work, we determined the structure of Klebsiella phage KP34p57 capsular depolymerase and dissected the role of individual domains in trimerization and functional activity. The crystal structure serendipitously revealed that the enzyme can exist in a monomeric state once deprived of its C-terminal domain. Based on the crystal structure and site-directed mutagenesis, we localized the key catalytic residues in an intra-subunit deep groove. Consistently, we show that C-terminally trimmed KP34p57 variants are monomeric, stable, and fully active. The elaboration of monomeric, fully active phage depolymerases is innovative in the field, as no previous example exists. Indeed, mini phage depolymerases can be combined in chimeric enzymes to extend their activity ranges, allowing their use against multiple serotypes.

Virulence Factors in Mycobacterium tuberculosis Infection: Structural and Functional Studies.

Tuberculosis (TB) remains one of the main causes of death by infection, especially in immunocompromised patients [...].

Female Sex Determination Factors in Ceratitis capitata: Molecular and Structural Basis of TRA and TRA2 Recognition.

In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. Here, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. We also generated a three-dimensional model of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers.

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.

The Oncosuppressive Properties of KCTD1: Its Role in Cell Growth and Mobility.

The KCTD protein family is traditionally regarded as proteins that play key roles in neurological physiopathology. However, new studies are increasingly demonstrating their involvement in many other biological processes, including cancers. This is particularly evident for KCTD proteins not involved in protein ubiquitination and degradation, such as KCTD1. We explored the role of KCTD1 in colorectal cancer by knocking down this protein in the human colon adenocarcinoma cell line, SW480. We re-assessed its ability to downregulate ß-catenin, a central actor in the WNT/ß-catenin signalling pathway. Interestingly, opposite effects are observed when the protein is upregulated in CACO2 colorectal cancer cells. Moreover, interrogation of the TCGA database indicates that KCTD1 downregulation is associated with ß-catenin overexpression in colorectal cancer patients. Indeed, knocking down KCTD1 in SW480 cells led to a significant increase in their motility and stemness, two important tumorigenesis traits, suggesting an oncosuppressor role for KCTD1. It is worth noting that similar effects are induced on colorectal cancer cells by the misregulation of KCTD12, a protein that is distantly related to KCTD1. The presented results further expand the spectrum of KCTD1 involvement in apparently unrelated physiopathological processes. The similar effects produced on colorectal cancer cell lines by KCTD1 and KCTD12 suggest novel, previously unreported analogous activities among members of the KCTD protein family.

A Structural View at Vaccine Development against M. tuberculosis.

Tuberculosis (TB) is still the leading global cause of death from an infectious bacterial agent. Limiting tuberculosis epidemic spread is therefore an urgent global public health priority. As stated by the WHO, to stop the spread of the disease we need a new vaccine, with better coverage than the current Mycobacterium bovis BCG vaccine. This vaccine was first used in 1921 and, since then, there are still no new licensed tuberculosis vaccines. However, there is extremely active research in the field, with a steep acceleration in the past decades, due to the advance of technologies and more rational vaccine design strategies. This review aims to gather latest updates in vaccine development in the various clinical phases and to underline the contribution of Structural Vaccinology (SV) to the development of safer and effective antigens. In particular, SV and the development of vaccine adjuvants is making the use of subunit vaccines, which are the safest albeit the less antigenic ones, an achievable goal. Indeed, subunit vaccines overcome safety concerns but need to be rationally re-engineered to enhance their immunostimulating effects. The larger availability of antigen structural information as well as a better understanding of the complex host immune response to TB infection is a strong premise for a further acceleration of TB vaccine development.

Management of periprosthetic knee joint infections: focus on the role of Nuclear Medicine (v2).

Background: When faced with a painful knee replacement, ruling out infection is mandatory to set the correct therapeutic approach. However, it is not always easy, especially in subclinical/chronic infections. A multidisciplinary approach is necessary to assess in the most correct way each case of suspected periprosthetic knee joint infection. This review explores the role of nuclear medicine investigations in the management of periprosthetic knee infections and their proper use within a multidisciplinary pathway. Methods: A PubMed search was conducted selecting studies from the past 10 years. Results: Triphasic bone scintigraphy has high sensitivity (93%) but poor specificity (56%) for periprosthetic joint infections of the knee, with a high negative predictive value (NPV), ranging from 96% to 100%. Consequently, a negative bone scan is useful in ruling out infection. In contrast, radiolabeled leukocyte scintigraphy is characterized by a sensitivity of 85.7-93%, specificity of 93.6-100%, diagnostic accuracy of 92.6-98%, NPV of 93-97.8%, and positive predictive value (PPV) of 66.7-100%. By adding a tomographic acquisition with hybrid single-photon emission computed tomography combined with computed tomography technique (SPECT/CT), the diagnostic accuracy increases. Because 18F-fluorodeoxyglucose (FDG) accumulates at both sites of inflammation and infection, FDG positron emission tomography (PET/CT) shows low specificity. Conclusions: A common decision-making process in the diagnosis of periprosthetic joint infection is not yet validated and multidisciplinary integration is mandatory. In this context, nuclear medicine can contribute decisively.

Structure based design of effective HtpG-derived vaccine antigens against M. tuberculosis.

Vaccine development against Tuberculosis is a strong need, given the low efficacy of the sole vaccine hitherto used, the Bacillus Calmette-Guérin (BCG) vaccine. The chaperone-like protein HtpGMtb of M. tuberculosis is a large dimeric and multi-domain protein with promising antigenic properties. We here used biophysical and biochemical studies to improve our understanding of the structural basis of HtpGMtb functional role and immunogenicity, a precious information to engineer improved antigens. We showed that HtpGMtb is a dimeric nucleotide-binding protein and identified the dimerisation interface on the C-terminal domain of the protein. We also showed that the most immunoreactive regions of the molecule are located on the C-terminal and middle domains of the protein, whereas no role is played by the catalytic N-terminal domain in the elicitation of the immune response. Based on these observations, we experimentally validated our predictions in mice, using a plethora of immunological assays. As an outcome, we designed vaccine antigens with enhanced biophysical properties and ease of production, albeit conserved or enhanced antigenic properties. Our results prove the efficacy of structural vaccinology approaches in improving our understanding of the structural basis of immunogenicity, a precious information to engineer more stable, homogeneous, efficiently produced, and effective vaccine antigens.

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.

Structural and Binding Properties of the Active Cell Wall Hydrolase RipA from M. tuberculosis, a Promising Biosensing Molecule for Early Warning Bacterial Detection.

BACKGROUND: Peptidoglycan is an essential component of the cell wall in all bacteria. In particular, the cell walls of Gram-positive bacteria are composed mostly of a thick layer of peptidoglycan. Its accessibility has important implications for their sensing in whole bacterial detection methodologies. Indeed, there is an urgent demand for rapid tests which can identify whole bacteria, e.g., directly at the point of care. OBJECTIVE: The aim of this work is to explore the suitability of RipA, a key cell division protein of M. tuberculosis, for whole cell biosensing of Gram-positive bacteria. METHODS: We here conducted Molecular Dynamics (MD) studies aimed at the understanding of the structural and dynamic features of active RipA and at the design of a suitable bioreceptor. Based on these studies, we engineered a RipA variant for covalent oriented immobilisation on golden surfaces and are able to bind peptidoglycan, albeit without degrading it. Surface Plasmon Resonance (SPR) was employed to check the ability of functionalized golden chips to recognize whole bacteria. RESULTS: MD analyses elucidated the structural details of the active form of RipA and suggested that this enzyme, once inactivated, presents a rigid and well-exposed peptidoglycan recognition cleft. We engineered RipA for proper oriented immobilisation on golden chips for SPR studies. Results show that once chemically coupled to a golden chip, the developed RipA-based bioreceptor is able to detect B. subtilis, used as a model in a concentration-dependent mode. CONCLUSION: Results highlight the potential of the engineered molecule to be integrated in the development of early warning biosensors for Gram-positive contamination in clinical diagnosis or food-borne infections.

AlphaFold-Predicted Structures of KCTD Proteins Unravel Previously Undetected Relationships among the Members of the Family.

One of the most striking features of KCTD proteins is their involvement in apparently unrelated yet fundamental physio-pathological processes. Unfortunately, comprehensive structure-function relationships for this protein family have been hampered by the scarcity of the structural data available. This scenario is rapidly changing due to the release of the protein three-dimensional models predicted by AlphaFold (AF). Here, we exploited the structural information contained in the AF database to gain insights into the relationships among the members of the KCTD family with the aim of facilitating the definition of the structural and molecular basis of key roles that these proteins play in many biological processes. The most important finding that emerged from this investigation is the discovery that, in addition to the BTB domain, the vast majority of these proteins also share a structurally similar domain in the C-terminal region despite the absence of general sequence similarities detectable in this region. Using this domain as reference, we generated a novel and comprehensive structure-based pseudo-phylogenetic tree that unraveled previously undetected similarities among the protein family. In particular, we generated a new clustering of the KCTD proteins that will represent a solid ground for interpreting their many functions.

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.

A structure-based approach for the development of a bicyclic peptide acting as a miniaturized anti-CD55 antibody.

CD55 is a major regulator of the complement system, a complex network of proteins that cooperate to clear tissue and blood pathogens from the organism. Indeed, overexpression of CD55 is associated with many diseases and is connected to the resistance mechanisms exhibited by several cancers towards immunotherapy approaches. High level of CD55 expression on tumour cells renders it a good target for both imaging and immunotherapy. Indeed, a conceivable approach to tackle disease is to interfere with CD55-mediated complement regulation with the use of CD55-targeting antibodies. However, the large size and poor tissue penetration together with to the high costs of antibodies often limits their widespread therapeutic use. Here, we employed bioinformatic and chemical approaches to design and synthesize molecules of small dimensions able to mimic a CD55 blocking antibody. As a result, a bicyclic peptide, named as miniAB55, proved to bind CD55 with nanomolar affinity. This molecule represents an attracting chemical scaffold for CD55-directed diagnostic tools in diseases associated with CD55 overproduction. To further support the applicative potential of miniAB55, we prove that the miniAB55 binds CD55 on the same region involved in inactivation of the complement C3 and C5 convertases, thus opening promising scenarios for the development of complement-modulating tools.

Host DDX Helicases as Possible SARS-CoV-2 Proviral Factors: A Structural Overview of Their Hijacking Through Multiple Viral Proteins.

As intracellular parasites, viruses hijack the host cell metabolic machinery for their replication. Among other cellular proteins, the DEAD-box (DDX) RNA helicases have been shown to be hijacked by coronaviruses and to participate in essential DDX-mediated viral replication steps. Human DDX RNA helicases play essential roles in a broad array of biological processes and serve multiple roles at the virus-host interface. The viral proteins responsible for DDX interactions are highly conserved among coronaviruses, suggesting that they might also play conserved functions in the SARS-CoV-2 replication cycle. In this review, we provide an update of the structural and functional data of DDX as possible key factors involved in SARS-CoV-2 hijacking mechanisms. We also attempt to fill the existing gaps in the available structural information through homology modeling. Based on this information, we propose possible paths exploited by the virus to replicate more efficiently by taking advantage of host DDX proteins. As a general rule, sequestration of DDX helicases by SARS-CoV-2 is expected to play a pro-viral role in two ways: by enhancing key steps of the virus life cycle and, at the same time, by suppressing the host innate immune response.

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.

Guanidinium binding to proteins: The intriguing effects on the D1 and D2 domains of Thermotoga maritima Arginine Binding Protein and a comprehensive analysis of the Protein Data Bank.

Thermotoga maritima Arginine Binding Protein has been extensively characterized because of its peculiar features and its possible use as a biosensor. In this characterization, deletion of the C-terminal helix to obtain the monomeric protein TmArgBP20-233 and dissection of the monomer in its two domains, D1 and D2, have been performed. In the present study the stability of these three forms against guanidinium chloride is investigated by means of circular dichroism and differential scanning calorimetry measurements. All three proteins show a high conformational stability; moreover, D1 shows an unusual behavior in the presence of low concentrations of guanidinium chloride. This finding has led us to investigate a possible binding interaction by means of isothermal titration calorimetry and X-ray crystallography; the results indicate that D1 is able to bind the guanidinium ion (GuH+), due to its similarity with the arginine terminal moiety. The analysis of the structural and dynamic properties of the D1-GuH+ complex indicates that the protein binds the ligand through multiple and diversified interactions. An exhaustive survey of the binding modes of GuH+ to proteins indicates that this is a rather common feature. These observations provide interesting insights into the effects that GuH+ is able to induce in protein structures.

A Structural View of SARS-CoV-2 RNA Replication Machinery: RNA Synthesis, Proofreading and Final Capping.

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.

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.

Structural features of HtpGMtb and HtpG-ESAT6Mtb vaccine antigens against tuberculosis: Molecular determinants of antigenic synergy and cytotoxicity modulation.

Vaccine development against tuberculosis is an urgent need as the only available vaccine, M. bovis Bacillus Calmette Guerin (BCG), is unable to provide significant protection in adults. Among newly identified antigens, Rv2299c is an excellent candidate for the rational design of an effective multi-antigenic TB vaccine. Also, when fused to the T cell antigen ESAT6, it becomes highly effective in boosting BCG immunization and it adopts low cytotoxicity compared to ESAT6. We here characterize these proteins by coupling various biophysical techniques to cytofluorimetry and computational studies. Altogether, our data provide an experimental evidence of the role of Rv2299c as a dimeric and highly thermostable molecular chaperone, here denoted as HtpGMtb. Molecular dynamics simulations show that ATP rigidly anchors the ATP-binding loop in a conformation incompatible with the structure of the free enzyme. We also show that HtpGMtb dimeric state is an important molecular feature for the improved antigenic and cytotoxic properties of HtpG-ESAT6Mtb. Indeed, structural features of HtpG-ESAT6Mtb show that not only does this molecule combine the antigenic properties of HtpGMtb and ESAT6, but HtpGMtb locks ESAT6 in a dimeric state, thus improving its cytotoxicity properties. The data presented here provide solid basis for the rational design of upgraded antigens.