Fibrillar Collagen Trimerization: Structural Basis and Related Genetic Disorders

The C-propeptides of fibrillar procollagens play crucial roles in tissue homeostasis and remodeling by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in the C-propeptides affecting molecular assembly are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Cells often produce multiple collagen types, each with the correct chain composition. In fibrillar collagens, molecular assembly begins with the C-propeptides which contain chain recognition sequences specific for each collagen type. Our recent crystal structure of a C-propeptide trimer from procollagen III (Bourhis et al, 2012), revealed specific interactions at the trimer interface. Unlike collagen III, a homotrimer, collagen I is normally a heterotrimer, though small amounts of homotrimer are found in embryonic tissue and cancer. To investigate the molecular basis of homo- versus hetero-trimer formation, further structural information is required. We have initiated structural studies on homo- and hetero-trimers of the C-propeptide domain of human procollagen I, to study the molecular basis of chain selectivity within the same cells. CPI homotrimer crystallizes in the monoclinic spacegroup P21, and data were collected to 2.2 Å resolution. The crystal structure solved by MR shows a structure resembling CPIII with the overall shape of a flower. At the trimerization interface however, interactions between chains are specific to CPI and these give insights into the mechanism of molecular recognition. These interactions will be compared to those in CPIII. Structural mapping indicates striking correlations between the sites of numerous disease-related mutations in different C-propeptide domains and the degree of phenotype severity. The results have broad implications for the understanding of genetic disorders of connective tissues and also for new therapeutic approaches against fibrosis.

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Structural basis for the influence of a single mutation K145N on the oligomerization and photoswitching rate of Dronpa

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444912039686 ◽

2012 ◽

Vol 68 (12) ◽

pp. 1653-1659 ◽

Cited By ~ 6

Author(s):

Ngan Nguyen Bich ◽

Benjamien Moeyaert ◽

Kristof Van Hecke ◽

Peter Dedecker ◽

Hideaki Mizuno ◽

...

Keyword(s):

Crystal Structure ◽

Optical Microscopy ◽

Molecular Basis ◽

Fluorescent Protein ◽

Image Resolution ◽

Structural Basis ◽

Single Mutation ◽

Switching Rate ◽

Absorption And Emission ◽

Emission Properties

The crystal structure of the on-state of PDM1-4, a single-mutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 Å resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain β-strands caused by oligomerization along theACinterface.

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Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines

Nature Communications ◽

10.1038/s41467-020-20478-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Manmohan Sharma ◽

Nipun Malhotra ◽

Manickam Yogavel ◽

Karl Harlos ◽

Bruno Melillo ◽

...

Keyword(s):

Crystal Structure ◽

Protein Synthesis ◽

Single Dose ◽

Molecular Basis ◽

Malaria Parasite ◽

Trna Synthetase ◽

Structural Basis ◽

Atp Binding Site ◽

Structural Framework ◽

Competitive Inhibitors

AbstractThe inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.

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Structural basis for regiospecific midazolam oxidation by human cytochrome P450 3A4

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616198114 ◽

2016 ◽

Vol 114 (3) ◽

pp. 486-491 ◽

Cited By ~ 43

Author(s):

Irina F. Sevrioukova ◽

Thomas L. Poulos

Keyword(s):

Crystal Structure ◽

Cytochrome P450 ◽

Active Site ◽

Structural Information ◽

Binding Mode ◽

Structural Basis ◽

Cytochrome P450 3A4 ◽

Human Cytochrome ◽

Cyp3a4 Substrate

Human cytochrome P450 3A4 (CYP3A4) is a major hepatic and intestinal enzyme that oxidizes more than 60% of administered therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the active site to regioselectively oxidize chemically diverse compounds is critical for better understanding structure–function relations in this important enzyme, improving the outcomes for drug metabolism predictions, and developing pharmaceuticals that have a decreased ability to undergo metabolism and cause detrimental drug–drug interactions. However, there is very limited structural information on CYP3A4–substrate interactions available to date. Despite the vast variety of drugs undergoing metabolism, only the sedative midazolam (MDZ) serves as a marker substrate for the in vivo activity assessment because it is preferentially and regioselectively oxidized by CYP3A4. We solved the 2.7 Å crystal structure of the CYP3A4–MDZ complex, where the drug is well defined and oriented suitably for hydroxylation of the C1 atom, the major site of metabolism. This binding mode requires H-bonding to Ser119 and a dramatic conformational switch in the F–G fragment, which transmits to the adjacent D, E, H, and I helices, resulting in a collapse of the active site cavity and MDZ immobilization. In addition to providing insights on the substrate-triggered active site reshaping (an induced fit), the crystal structure explains the accumulated experimental results, identifies possible effector binding sites, and suggests why MDZ is predominantly metabolized by the CYP3A enzyme subfamily.

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Structural basis for the evolution of vancomycin resistance D,D-peptidases

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314092845 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C715-C715

Author(s):

Peter Stogios ◽

Djalal Meziane-Cherif ◽

Elena Evdokimova ◽

Patrice Courvalin ◽

Alexei Savchenko

Keyword(s):

Molecular Basis ◽

Structural Information ◽

Function Analysis ◽

Structural Basis ◽

Binding Affinities ◽

Structural Elements ◽

Structural Element ◽

Enterococcus Spp ◽

Extended Cavity ◽

High Level

Emergence of high-level resistance to the last resort glycopeptide antibiotic vancomycin in Enterococcus spp. and its spread to methicillin-resistant Staphylococcus aureus is a public health threat. Resistance to vancomycin is due to substitution of the D-Ala-D-Ala terminus of cell wall precursors, which forms the antibiotic target, by D-Ala-D-Lac or D-Ala-D-Ser of low binding affinities. Resistance also requires depletion of the normal precursors catalyzed by the zinc-dependent D,D-peptidases VanX and VanY acting on dipeptide (D-Ala-D-Ala) or pentapeptide (UDP-MurNac-L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala), respectively. Some resistance operons encode VanXY D,D-peptidase acting on both substrates. Van D,D-peptidases represent attractive targets for combinational antimicrobial therapies to curb resistance; however, the molecular basis of their specificity remains poorly understood, hindering development of potent inhibitors. Therefore we undertook detailed structure-function analysis of VanXY and VanY enzymes. Obtained structural information revealed the substrate-binding site of VanXYC as an extended cavity suitable for binding of di- or pentapeptides, contrasting with previous results showing that VanX contains a small, shallow active site. Biochemical and mutagenesis analysis identified a mobile cap over the catalytic site of VanXYC as the key structural element involved in a switch between di- and pentapeptide hydrolysis. The structures also provided the molecular basis for selectivity towards Van-susceptible peptidoglycan precursors. Overall, this study illustrates the adaptability of the D,D-peptidase fold in response to antibiotic pressure via evolution of particular structural elements that modulate substrate specificity. The results open new opportunities for structure-guided development of Van D,D-peptidases specific inhibitors as glycopeptides adjuvants. This project has been funded by NIAID under Contracts No. HHSN272200700058C and HHSN272201200026C.

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Crystal structure of human cytoplasmic tRNAHis-specific 5′-monomethylphosphate capping enzyme

Nucleic Acids Research ◽

10.1093/nar/gkz1216 ◽

2020 ◽

Vol 48 (3) ◽

pp. 1572-1582 ◽

Cited By ~ 1

Author(s):

Yining Liu ◽

Anna Martinez ◽

Seisuke Yamashita ◽

Kozo Tomita

Keyword(s):

Crystal Structure ◽

Molecular Basis ◽

Poor Prognosis ◽

Structural Model ◽

Structural Information ◽

Breast Cancers ◽

Methyl Group ◽

Capping Enzyme ◽

Trna Species ◽

Α Helix

Abstract BCDIN3 domain containing RNA methyltransferase, BCDIN3D, monomethylates the 5′-monophosphate of cytoplasmic tRNAHis with a G−1:A73 mispair at the top of an eight-nucleotide-long acceptor helix, using S-adenosyl-l-methionine (SAM) as a methyl group donor. In humans, BCDIN3D overexpression is associated with the tumorigenic phenotype and poor prognosis in breast cancer. Here, we present the crystal structure of human BCDIN3D complexed with S-adenosyl-l-homocysteine. BCDIN3D adopts a classical Rossmann-fold methyltransferase structure. A comparison of the structure with that of the closely related methylphosphate capping enzyme, MePCE, which monomethylates the 5′-γ-phosphate of 7SK RNA, revealed the important residues for monomethyl transfer from SAM onto the 5′-monophosphate of tRNAHis and for tRNAHis recognition by BCDIN3D. A structural model of tRNAHis docking onto BCDIN3D suggested the molecular mechanism underlying the different activities between BCDIN3D and MePCE. A loop in BCDIN3D is shorter, as compared to the corresponding region that forms an α-helix to recognize the 5′-end of RNA in MePCE, and the G−1:A73 mispair in tRNAHis allows the N-terminal α-helix of BCDIN3D to wedge the G−1:A73 mispair of tRNAHis. As a result, the 5′-monophosphate of G−1 of tRNAHis is deep in the catalytic pocket for 5′-phosphate methylation. Thus, BCDIN3D is a tRNAHis-specific 5′-monomethylphosphate capping enzyme that discriminates tRNAHis from other tRNA species, and the structural information presented in this study also provides the molecular basis for the development of drugs against breast cancers.

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Structural Basis for Effector Control and Redox Partner Recognition in Cytochrome P450

Science ◽

10.1126/science.1235797 ◽

2013 ◽

Vol 340 (6137) ◽

pp. 1227-1230 ◽

Cited By ~ 116

Author(s):

Sarvind Tripathi ◽

Huiying Li ◽

Thomas L. Poulos

Keyword(s):

Crystal Structure ◽

Electron Transfer ◽

Structural Information ◽

Cytochromes P450 ◽

Structural Basis ◽

Proton Coupled Electron Transfer ◽

Redox Partner ◽

Open Conformation ◽

Redox Partners

Cytochromes P450 catalyze a variety of monooxygenase reactions that require electron transfer from redox partners. Although the structure of many P450s and a small handful of redox partners are known, there is very little structural information available on redox complexes, thus leaving a gap in our understanding on the control of P450–redox partner interactions. We have solved the crystal structure of oxidized and reduced P450cam complexed with its redox partner, putidaredoxin (Pdx), to 2.2 and 2.09 angstroms, respectively. It was anticipated that Pdx would favor closed substrate-bound P450cam, which differs substantially from the open conformer, but instead we found that Pdx favors the open state. These new structures indicate that the effector role of Pdx is to shift P450cam toward the open conformation, which enables the establishment of a water-mediated H-bonded network, which is required for proton-coupled electron transfer.

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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