Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism

The X-ray crystal structure of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae (HiDapE) bound by the products of hydrolysis, succinic acid and l,l-DAP, was determined at 1.95 Å. Surprisingly, the structure bound to the products revealed that HiDapE undergoes a significant conformational change in which the catalytic domain rotates ∼50° and shifts ∼10.1 Å (as measured at the position of the Zn atoms) relative to the dimerization domain. This heretofore unobserved closed conformation revealed significant movements within the catalytic domain compared to that of wild-type HiDapE, which results in effectively closing off access to the dinuclear Zn(II) active site with the succinate carboxylate moiety bridging the dinculear Zn(II) cluster in a μ-1,3 fashion forming a bis(μ-carboxylato)dizinc(II) core with a Zn–Zn distance of 3.8 Å. Surprisingly, His194.B, which is located on the dimerization domain of the opposing chain ∼10.1 Å from the dinuclear Zn(II) active site, forms a hydrogen bond (2.9 Å) with the oxygen atom of succinic acid bound to Zn2, forming an oxyanion hole. As the closed structure forms upon substrate binding, the movement of His194.B by more than ∼10 Å is critical, based on site-directed mutagenesis data, for activation of the scissile carbonyl carbon of the substrate for nucleophilic attack by a hydroxide nucleophile. Employing the HiDapE product-bound structure as the starting point, a reverse engineering approach called product-based transition-state modeling provided structural models for each major catalytic step. These data provide insight into the catalytic reaction mechanism and also the future design of new, potent inhibitors of DapE enzymes

Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG

AbstractThe spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites

Corrigendum: Nα -acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Graphical Abstrac

Nα-acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express Nɑ -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N5, N5-di-methyl Nα-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104 M−1s−1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7 μM; Ki = 37.1 ± 0.85 μM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1 μM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 μM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn)

An integrated process for planning, delivery, and stewardship of urban nature-based solutions: the Connecting Nature Framework

Mainstreaming nature-based solutions in cities has grown in scale and magnitude in recent times but is still considered to be the main challenge for transitioning our cities and their communities to be more climate resilient and liveable: environmentally, economically, and socially. Furthermore, taking nature-based solutions to the next level, and scaling them out to all urban contexts to achieve a greater impact, is proving to be slow and often conflicts with other transitioning initiatives such as energy generation, mobility and transport initiatives, and infilling to combat sprawl. So, the task is neither easy nor straightforward; there are many barriers to this novel transition, especially when it comes to collaborative approaches to implementing nature-based solutions with diverse urban communities and within city authorities themselves. This paper reports on a new process that is systematically co-produced and captured as a framework for planning nature-based solutions that emerged during the Connecting Nature project. The Connecting Nature Framework is a three-stage, iterative process that involves seven key activity areas for mainstreaming nature-based solutions: technical solutions, governance, financing and business models, nature-based enterprises, co-production, reflexive monitoring, and impact assessment. The tested and applied framework is designed to address and overcome barriers to the implementation of nature-based solutions in cities via a co-created, iterative, and reflective approach. The planning process guided by the proposed framework has already yielded promising results with some of the cities of the project, though further usage and its adoption by other cities is needed to explore its potential in different contexts especially in the Global South. The paper concludes with suggestions on how this may be realised

Potrzeby kompetencyjne pracowników jednostek samorządu terytorialnego (JST)

Polish Abstract: Rola kompetencji pracowników jako kluczowego czynnika sukcesu współczesnych organizacji nie budzi już wątpliwości. W odniesieniu do sektora komercyjnego wiele uwagi poświęca się zarówno analizom teoretycznym, jak i praktycznym wysiłkom w zakresie podnoszenia poziomu kompetencji pracowniczych. Przedsiębiorcy rozumieją, iż bez ciągłego poszerzania wiedzy i umiejętności oraz kształtowania właściwych postaw swoich pracowników nie będą w stanie utrzymać konkurencyjnej pozycji na rynku. Znacznie mniej uwagi poświęca się kompetencjom pracowników sektora publicznego, czasem nietrafnie kojarząc je wyłącznie ze znajomością prawa i umiejętnościami przestrzegania procedur administracyjnych. Tymczasem organizacje sektora publicznego funkcjonują w dynamicznym otoczeniu, a społeczeństwo stawia przed nimi coraz bardziej wymagające cele. Jednostki samorządu terytorialnego (JST) mają nie tylko zapewniać proste usługi administracyjne, ale przede wszystkim ich misją ma być szeroko rozumiany proces rozwoju społeczno-gospodarczego na danym terenie. Sprostanie tym nowym wyzwaniom wymaga poświęcenia uwagi problematyce kompetencji pracowników JST. Niezbędne jest postrzeganie potrzeb kompetencyjnych pracowników samorządowych także w kontekście zadań związanych z rozwojem. Świadomość struktury potrzeb kompetencyjnych i aktualnego stanu kompetencji może być właściwą podstawą do podejmowania interwencji związanych z procesem szkoleń jako ważnym instrumentem doskonalenia polskiej administracji samorządowej. English Abstract: The role of employees' competences as a key factor in the success of modern organizations no longer raises any doubts. With regard to the commercial sector, much attention is paid to both theoretical analysis and practical efforts in raising the level of employee competences. Entrepreneurs understand that without constantly expanding knowledge and skills and shaping the right attitudes of their employees, they will not be able to maintain a competitive position on the market. Much less attention is paid to the competences of public sector employees, sometimes incorrectly associating them only with the knowledge of law and the ability to comply with administrative procedures. Meanwhile, public sector organizations operate in a dynamic environment, and society sets more and more demanding goals for them. Local government units (LGUs) are not only to provide simple administrative services, but above all their mission is to be broadly understood the process of socio-economic development in a given area. Meeting these new challenges requires devoting attention to the competences of LGU employees. It is necessary to perceive the competence needs of local government employees also in the context of tasks related to development. Awareness of the structure of competency needs and the current state of competencies may be the appropriate basis for undertaking interventions related to the training process as an important instrument for improving the Polish local government administration

Potrzeby kompetencyjne pracowników jednostek samorządu terytorialnego (JST)

Polish Abstract: Rola kompetencji pracowników jako kluczowego czynnika sukcesu współczesnych organizacji nie budzi już wątpliwości. W odniesieniu do sektora komercyjnego wiele uwagi poświęca się zarówno analizom teoretycznym, jak i praktycznym wysiłkom w zakresie podnoszenia poziomu kompetencji pracowniczych. Przedsiębiorcy rozumieją, iż bez ciągłego poszerzania wiedzy i umiejętności oraz kształtowania właściwych postaw swoich pracowników nie będą w stanie utrzymać konkurencyjnej pozycji na rynku. Znacznie mniej uwagi poświęca się kompetencjom pracowników sektora publicznego, czasem nietrafnie kojarząc je wyłącznie ze znajomością prawa i umiejętnościami przestrzegania procedur administracyjnych. Tymczasem organizacje sektora publicznego funkcjonują w dynamicznym otoczeniu, a społeczeństwo stawia przed nimi coraz bardziej wymagające cele. Jednostki samorządu terytorialnego (JST) mają nie tylko zapewniać proste usługi administracyjne, ale przede wszystkim ich misją ma być szeroko rozumiany proces rozwoju społeczno-gospodarczego na danym terenie. Sprostanie tym nowym wyzwaniom wymaga poświęcenia uwagi problematyce kompetencji pracowników JST. Niezbędne jest postrzeganie potrzeb kompetencyjnych pracowników samorządowych także w kontekście zadań związanych z rozwojem. Świadomość struktury potrzeb kompetencyjnych i aktualnego stanu kompetencji może być właściwą podstawą do podejmowania interwencji związanych z procesem szkoleń jako ważnym instrumentem doskonalenia polskiej administracji samorządowej. English Abstract: The role of employees' competences as a key factor in the success of modern organizations no longer raises any doubts. With regard to the commercial sector, much attention is paid to both theoretical analysis and practical efforts in raising the level of employee competences. Entrepreneurs understand that without constantly expanding knowledge and skills and shaping the right attitudes of their employees, they will not be able to maintain a competitive position on the market. Much less attention is paid to the competences of public sector employees, sometimes incorrectly associating them only with the knowledge of law and the ability to comply with administrative procedures. Meanwhile, public sector organizations operate in a dynamic environment, and society sets more and more demanding goals for them. Local government units (LGUs) are not only to provide simple administrative services, but above all their mission is to be broadly understood the process of socio-economic development in a given area. Meeting these new challenges requires devoting attention to the competences of LGU employees. It is necessary to perceive the competence needs of local government employees also in the context of tasks related to development. Awareness of the structure of competency needs and the current state of competencies may be the appropriate basis for undertaking interventions related to the training process as an important instrument for improving the Polish local government administration

Embedding co-production of nature-based solutions in urban governance: Emerging co-production capacities in three European cities

This paper seeks to understand how co-production can become embedded as a collaborative governance practice by which city governments plan, deliver and steward nature-based solutions. To these ends, the paper analyses how policy officers manifest capacities for co-production in three European cities – Genk (Belgium), Glasgow (United Kingdom) and Poznań (Poland) – while experimenting with co-production to develop and scale nature-based solutions. Co-production capacities include conditions and activities to (1) create space for co-production, (2) safeguard inclusive and legitimate co-production, and (3) link co-production processes and results to contexts. The results demonstrate how policy officers in the three cities have mobilised and created resources, skills, institutional support and partnerships to implement diverse processes to co-produce nature-based solutions. While these conditions mark starting changes in urban governance, engaging with and embedding co-production causes tensions between the dynamic and diffuse nature of co-production and existing formal governance settings and processes. Lessons for strengthening the capacities to embed co-production as a collaborative governance practice in nature-based solutions planning, delivery and stewardship are: (1) embedding a tailor-made approach for inclusive co-production to meaningfully engage diverse actors in place-based settings, (2) embedding open-ended co-production with long-term benefits, and (3) embedding new relations and roles to sustain co-production

Multiple, but Concerted Cellular Activities of the Human Protein Hap46/BAG-1M and Isoforms

The closely related human and murine proteins Hap46/BAG-1M and BAG-1, respectively, were discovered more than a decade ago by molecular cloning techniques. These and the larger isoform Hap50/BAG-1L, as well as shorter isoforms, have the ability to interact with a seemingly unlimited array of proteins of completely unrelated structures. This problem was partially resolved when it was realized that molecular chaperones of the hsp70 heat shock protein family are major primary association partners, binding being mediated by the carboxy terminal BAG-domain and the ATP-binding domain of hsp70 chaperones. The latter, in turn, can associate with an almost unlimited variety of proteins through their substrate-binding domains, so that ternary complexes may result. The protein folding activity of hsp70 chaperones is affected by interactions with Hap46/BAG-1M or isoforms. However, there also exist several proteins which bind to Hap46/BAG-1M and isoforms independent of hsp70 mediation. Moreover, Hap46/BAG-1M and Hap50/BAG-1L, but not the shorter isoforms, can bind to DNA in a sequence-independent manner by making use of positively charged regions close to their amino terminal ends. This is the molecular basis for their effects on transcription which are of major physiological relevance, as discussed here in terms of a model. The related proteins Hap50/BAG-1L and Hap46/BAG-1M may thus serve as molecular links between such diverse bioactivities as regulation of gene expression and protein quality control. These activities are coordinated and synergize in helping cells to cope with conditions of external stress. Moreover, they recently became markers for the aggressiveness of several cancer types

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1