Biochemical and structural characterization of alanine racemase from Bacillus anthracis (Ames)

<p>Abstract</p> <p>Background</p> <p><it>Bacillus anthracis </it>is the causative agent of anthrax and a potential bioterrorism threat. Here we report the biochemical and structural characterization of <it>B. anthracis </it>(Ames) alanine racemase (Alr_<it>Bax</it>), an essential enzyme in prokaryotes and a target for antimicrobial drug development. We also compare the native Alr_{<it>Bax </it>}structure to a recently reported structure of the same enzyme obtained through reductive lysine methylation.</p> <p>Results</p> <p><it>B. anthracis </it>has two open reading frames encoding for putative alanine racemases. We show that only one, <it>dal1</it>, is able to complement a D-alanine auxotrophic strain of <it>E. coli</it>. Purified Dal1, which we term Alr_<it>Bax</it>, is shown to be a dimer in solution by dynamic light scattering and has a V_maxfor racemization (L- to D-alanine) of 101 U/mg. The crystal structure of unmodified Alr_{<it>Bax </it>}is reported here to 1.95 Å resolution. Despite the overall similarity of the fold to other alanine racemases, Alr_{<it>Bax </it>}makes use of a chloride ion to position key active site residues for catalysis, a feature not yet observed for this enzyme in other species. Crystal contacts are more extensive in the methylated structure compared to the unmethylated structure.</p> <p>Conclusion</p> <p>The chloride ion in Alr_{<it>Bax </it>}is functioning effectively as a carbamylated lysine making it an integral and unique part of this structure. Despite differences in space group and crystal form, the two Alr_{<it>Bax </it>}structures are very similar, supporting the case that reductive methylation is a valid rescue strategy for proteins recalcitrant to crystallization, and does not, in this case, result in artifacts in the tertiary structure.</p

Structural basis of thiol-based regulation of formaldehyde detoxification in H. influenzae by a MerR regulator with no sensor region

Pathogenic bacteria such as Haemophilus influenzae, a major cause of lower respiratory tract diseases, must cope with a range of electrophiles generated in the host or by endogenous metabolism. Formaldehyde is one such compound that can irreversibly damage proteins and DNA through alkylation and cross-linking and interfere with redox homeostasis. Its detoxification operates under the control of HiNmlR, a protein from the MerR family that lacks a specific sensor region and does not bind metal ions. We demonstrate that HiNmlR is a thiol-dependent transcription factor that modulates H. influenzae response to formaldehyde, with two cysteine residues (Cys54 and Cys71) identified to be important for its response against a formaldehyde challenge. We obtained crystal structures of HiNmlR in both the DNA-free and two DNA-bound forms, which suggest that HiNmlR enhances target gene transcription by twisting of operator DNA sequences in a two-gene operon containing overlapping promoters. Our work provides the first structural insights into the mechanism of action of MerR regulators that lack sensor regions

Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations

The human genome encodes two active Vaccinia-related protein kinases (VRK), VRK1 and VRK2. These proteins have been implicated in a number of cellular processes and linked to a variety of tumors. However, understanding the cellular role of VRKs and establishing their potential use as targets for therapeutic intervention has been limited by the lack of tool compounds that can specifically modulate the activity of these kinases in cells. Here we identified BI-D1870, a dihydropteridine inhibitor of RSK kinases, as a promising starting point for the development of chemical probes targeting the active VRKs. We solved co-crystal structures of both VRK1 and VRK2 bound to BI-D1870 and of VRK1 bound to two broad-spectrum inhibitors. These structures revealed that both VRKs can adopt a P-loop folded conformation, which is stabilized by different mechanisms on each protein. Based on these structures, we suggest modifications to the dihydropteridine scaffold that can be explored to produce potent and specific inhibitors towards VRK1 and VRK2

Development of dihydropyrrolopyridinone-based PKN2/PRK2 chemical tools to enable drug discovery

The Protein Kinase N proteins (PKN1, PKN2 and PKN3) are Rho GTPase effectors. They are involved in several biological processes such as cytoskeleton organization, cell mobility, adhesion, and cell cycle. Recently PKNs have been reported as essential for survival in several tumor cell lines, including prostate and breast cancer. Here, we report the development of dihydropyrrolopyridinone-based inhibitors for PKN2 and its closest homologue, PKN1, and their associated structure–activity relationship (SAR). Our studies identified a range of molecules with high potency exemplified by compound 8 with Ki = 8 nM for PKN2 and 14x selectivity over PKN1. Membrane permeability and target engagement for PKN2 were assessed by a NanoBRET cellular assay. Importantly, good selectivity across the wider human kinome and other kinase family members was achieved. These compounds provide strong starting points for lead optimization to PKN1/2 development compounds

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background: The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily. Methodology: In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family. Conclusions: Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. Thes

SGC-CAMKK2-1: A Chemical Probe for CAMKK2

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2

Chemical Space Exploration of Oxetanes

This paper focuses on new derivatives bearing an oxetane group to extend accessible chemical space for further identification of kinase inhibitors. The ability to modulate kinase activity represents an important therapeutic strategy for the treatment of human illnesses. Known as a nonclassical isoster of the carbonyl group, due to its high polarity and great ability to function as an acceptor of hydrogen bond, oxetane seems to be an attractive and underexplored structural motif in medicinal chemistry

Raising the Bar(-seq) in Leishmania genetic screens

Our understanding of regulatory factors in Leishmania differentia- tion has long been restricted by the available genetic tools, but the availability of CRISPR/Cas9 has changed the landscape forever. Recently, Baker and Catta-Preta et al. applied Cas9 editing and kinome-wide bar-seq to dissect the function of 204 kinases in the Leishmania mexicana life cycle.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq: 424729/2018FAPESP: 2018/09948-

Structural Characterization of Maize SIRK1 Kinase Domain Reveals an Unusual Architecture of the Activation Segment

Kinases are primary regulators of plant metabolism and excellent targets for plant breeding. However, most kinases, including the abundant receptor-like kinases (RLK), have no assigned role. SIRK1 is a leucine-rich repeat receptor-like kinase (LRR-RLK), the largest family of RLK. In Arabidopsis thaliana, SIRK1 (AtSIRK1) is phosphorylated after sucrose is resupplied to sucrose-starved seedlings and it modulates the sugar response by phosphorylating several substrates. In maize, the ZmSIRK1 expression is altered in response to drought stress. In neither Arabidopsis nor in maize has the function of SIRK1 been completely elucidated. As a first step toward the biochemical characterization of ZmSIRK1, we obtained its recombinant kinase domain, demonstrated that it binds AMP-PNP, a non-hydrolysable ATP-analog, and solved the structure of ZmSIRK1- AMP-PNP co-crystal. The ZmSIRK1 crystal structure revealed a unique conformation for the activation segment. In an attempt to find inhibitors for ZmSIRK1, we screened a focused small molecule library and identified six compounds that stabilized ZmSIRK1 against thermal melt. ITC analysis confirmed that three of these compounds bound to ZmSIRK1 with low micromolar affinity. Solving the 3D structure of ZmSIRK1-AMP-PNP co-crystal provided information on the molecular mechanism of ZmSIRK1 activity. Furthermore, the identification of small molecules that bind this kinase can serve as initial backbone for development of new potent and selective ZmSIRK1 antagonists

Structural Characterization of Maize SIRK1 Kinase Domain Reveals an Unusual Architecture of the Activation Segment

Kinases are primary regulators of plant metabolism and excellent targets for plant breeding. However, most kinases, including the abundant receptor-like kinases (RLK), have no assigned role. SIRK1 is a leucine-rich repeat receptor-like kinase (LRR-RLK), the largest family of RLK. In Arabidopsis thaliana, SIRK1 (AtSIRK1) is phosphorylated after sucrose is resupplied to sucrose-starved seedlings and it modulates the sugar response by phosphorylating several substrates. In maize, the ZmSIRK1 expression is altered in response to drought stress. In neither Arabidopsis nor in maize has the function of SIRK1 been completely elucidated. As a first step toward the biochemical characterization of ZmSIRK1, we obtained its recombinant kinase domain, demonstrated that it binds AMP-PNP, a non-hydrolysable ATP-analog, and solved the structure of ZmSIRK1- AMP-PNP co-crystal. The ZmSIRK1 crystal structure revealed a unique conformation for the activation segment. In an attempt to find inhibitors for ZmSIRK1, we screened a focused small molecule library and identified six compounds that stabilized ZmSIRK1 against thermal melt. ITC analysis confirmed that three of these compounds bound to ZmSIRK1 with low micromolar affinity. Solving the 3D structure of ZmSIRK1-AMP-PNP co-crystal provided information on the molecular mechanism of ZmSIRK1 activity. Furthermore, the identification of small molecules that bind this kinase can serve as initial backbone for development of new potent and selective ZmSIRK1 antagonists