Cryo-EM structure of human eIF5A-DHS complex reveals the molecular basis of hypusination-associated neurodegenerative disorders.

Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.

Structural analysis of new compound heterozygous variants in PEPD gene identified in a patient with Prolidase Deficiency diagnosed by exome sequencing.

Prolidase Deficiency (PD) is an autosomal recessive rare disorder caused by loss or reduction of prolidase enzymatic activity due to variants in the PEPD gene. PD clinical features vary among affected individuals: skin ulcerations, recurrent infections, and developmental delay are common. In this study, we describe a 16-year-old boy with a mild PD phenotype comprising chronic eczema, recurrent infections and elevated IgE. Whole exome sequencing analysis revealed three PEPD variants: c.575T>C p.(Leu192Pro) inherited from the mother, and c.692_694del p.(Tyr231del) and c.1409G>A p.(Arg470His), both inherited from the father. The variant p.(Tyr231del) has been previously characterized by high-resolution X-ray structure analysis as altering protein dynamics/flexibility. In order to study the effects of the other two prolidase variants, we performed site directed mutagenesis purification and crystallization studies. A high-resolution X-ray structure could only be obtained for the p.(Arg470His) variant, which showed no significant structural differences in comparison to WT prolidase. On the other hand, the p.(Leu192Pro) variant led to significant protein destabilization. Hence, we conclude that the maternal p.(Leu192Pro) variant was likely causally associated with the proband´s disease, together with the known pathogenic paternal variant p.(Tyr231del). Our results demonstrated the utility of exome sequencing to perform diagnosis in PD cases with mild phenotype.

Prolidase - A protein with many faces.

Prolidase is a metal-dependent peptidase specialized in the cleavage of dipeptides containing proline or hydroxyproline on their C-termini. Prolidase homologues are found in all kingdoms of life. The importance of prolidase in human health is underlined by a rare hereditary syndrome referred to as Prolidase Deficiency. A growing number of studies highlight the importance of prolidase in various other human conditions, including cancer. Some recent studies link prolidase's activity-independent regulatory role to tumorigenesis. Furthermore, the enzyme or engineered variants have some applications in biotechnology. In this short review, we aim to highlight different aspects of the protein the importance of which is increasingly recognized over the last years.

Structural Characterization of Glycerol Kinase from the Thermophilic Fungus Chaetomium thermophilum.

Glycerol is an organic compound that can be utilized as an alternative source of carbon by various organisms. One of the ways to assimilate glycerol by the cell is the phosphorylative catabolic pathway in which its activation is catalyzed by glycerol kinase (GK) and glycerol-3-phosphate (G3P) is formed. To date, several GK crystal structures from bacteria, archaea, and unicellular eukaryotic parasites have been solved. Herein, we present a series of crystal structures of GK from Chaetomium thermophilum (CtGK) in apo and glycerol-bound forms. In addition, we show the feasibility of an ADP-dependent glucokinase (ADPGK)-coupled enzymatic assay to measure the CtGK activity. New structures described in our work provide structural insights into the GK catalyzed reaction in the filamentous fungus and set the foundation for understanding the glycerol metabolism in eukaryotes.

Co-expression with chaperones can affect protein 3D structure as exemplified by loss-of-function variants of human prolidase.

Prolidase catalyzes the cleavage of dipeptides containing proline on their C terminus. The reduction in prolidase activity is the cause of a rare disease named 'Prolidase Deficiency'. Local structural disorder was indicated as one of the causes for diminished prolidase activity. Previous studies showed that heat shock proteins can partially recover prolidase activity in vivo. To analyze this mechanism of enzymatic activity rescue, we compared the crystal structures of selected prolidase mutants expressed in the absence and in the presence of chaperones. Our results confirm that protein chaperones facilitate the formation of more ordered structures by their substrate protein. These results also suggest that the protein expression system needs to be considered as an important parameter in structural studies. DATABASES: The reported crystal structures and their associated structure factor amplitudes were deposited in the Protein Data Bank under the accession codes 6SRE, 6SRF, and 6SRG, respectively.

Half Way to Hypusine-Structural Basis for Substrate Recognition by Human Deoxyhypusine Synthase.

Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved "ball-and-chain" motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.

Crystal Structure of Kluyveromyces lactis Glucokinase (KlGlk1).

Glucose phosphorylating enzymes are crucial in the regulation of basic cellular processes, including metabolism and gene expression. Glucokinases and hexokinases provide a pool of phosphorylated glucose in an adenosine diphosphate (ADP)- and ATP-dependent manner to shape the cell metabolism. The glucose processing enzymes from Kluyveromyces lactis are poorly characterized despite the emerging contribution of this yeast strain to industrial and laboratory scale biotechnology. The first reports on K. lactis glucokinase (KlGlk1) positioned the enzyme as an essential component required for glucose signaling. Nevertheless, no biochemical and structural information was available until now. Here, we present the first crystal structure of KlGlk1 together with biochemical characterization, including substrate specificity and enzyme kinetics. Additionally, comparative analysis of the presented structure and the prior structures of lactis hexokinase (KlHxk1) demonstrates the potential transitions between open and closed enzyme conformations upon ligand binding.