Molecular recognition at septin interfaces: the switches hold the key

ABSTRACTThe assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbours. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita’s postulate which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.HIGHLIGHTSHigh resolution structures of septin heterodimeric complexes reveal new interactionsSwitches of small GTPases are repurposed in septins to play key roles in interface contactsThe GTP present in catalytically inactive septins participates in molecular recognitionConservation of interface residues allows for subunit exchangeability from within septin subgroupsSpecific residues for each septin subgroup provide selectivity for proper filament assemblyGRAPHICAL ABSTRACT

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Molecular recognition of sugar binding in a melibiose transporter MelB by X-ray crystallography

10.1101/2020.12.20.423627 ◽

2020 ◽

Author(s):

Lan Guan ◽

Parameswaran Hariharan

Keyword(s):

Molecular Recognition ◽

Binding Site ◽

Crystal Structures ◽

Mammalian Cells ◽

Substrate Binding ◽

Structural Basis ◽

Cooperative Binding ◽

Recognition Mechanism ◽

Substrate Binding Site ◽

X Ray

AbstractThe symporter melibiose permease MelB is the best-studied representative from MFS_2 family and the only protein in this large family with crystal structure determined. Previous thermodynamic studies show that MelB utilizes a cooperative binding as the core mechanism for its obligatory symport. Here we present two sugar-bound X-ray crystal structures of a Salmonella typhimurium MelB D59C uniport mutant that binds and catalyzes melibiose transport uncoupled to either cation, as determined by biochemical and biophysical characterizations. The two structures with bound nitrophenyl-α-D-galactoside or dodecyl-β-D-melibioside, which were refined to a resolution of 3.05 or 3.15 Å, respectively, are virtually identical at an outward-facing conformation; each one contains a α-galactoside molecule in the middle of protein. In the substrate-binding site, the galactosyl moiety on both ligands are at an essentially same configuration, so a galactoside specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for the binding of sugar in MelB is deciphered. The data also allow to assign the conserved cation-binding pocket, which is directly connected to the sugar specificity determinant pocket. The intimate connection between the two selection sites lays the structural basis for the cooperative binding and coupled transport. This key structural finding answered the long-standing question on the substrate binding for the Na+-coupled MFS family of transporters.SignificanceMajor facilitator superfamily_2 transporters contain >10,000 members that are widely expressed from bacteria to mammalian cells, and catalyze uptake of varied nutrients from sugars to phospholipids. While several crystal structures with bound sugar for other MFS permeases have been determined, they are either uniporters or symporters coupled solely to H+. MelB catalyzes melibiose symport with either Na+, Li+, or H+, a prototype for Na+-coupled MFS transporters, but its sugar recognition has been a long-unsolved puzzle. Two high-resolution crystal structures presented here clearly reveal the molecular recognition mechanism for the binding of sugar in MelB. The substrate-binding site is characterized with a small specificity groove adjoining a large nonspecific cavity, which could offer a potential for future exploration of active transporters for drug delivery.

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High-Resolution Crystal Structures of Human Hemoglobin with Mutations at Tryptophan 37β: Structural Basis for a High-Affinity T-State†,‡

Biochemistry ◽

10.1021/bi9708702 ◽

1998 ◽

Vol 37 (13) ◽

pp. 4358-4373 ◽

Cited By ~ 25

Author(s):

Jeffrey S. Kavanaugh ◽

Jamie A. Weydert ◽

Paul H. Rogers ◽

Arthur Arnone

Keyword(s):

High Resolution ◽

Crystal Structures ◽

Structural Basis ◽

Human Hemoglobin ◽

High Affinity ◽

T State

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The structure and properties of septin 3: a possible missing link in septin filament formation

Biochemical Journal ◽

10.1042/bj20120851 ◽

2013 ◽

Vol 450 (1) ◽

pp. 95-105 ◽

Cited By ~ 28

Author(s):

Joci N. A. Macedo ◽

Napoleão F. Valadares ◽

Ivo A. Marques ◽

Frederico M. Ferreira ◽

Julio C. P. Damalio ◽

...

Keyword(s):

Crystal Structure ◽

Sequence Similarity ◽

Three Dimensional ◽

Building Blocks ◽

Structural Basis ◽

Filament Assembly ◽

Gtp Binding ◽

Hydrolysis Of ◽

Free Monomer ◽

Septin Filament

The human genome codes for 13 members of a family of filament-forming GTP-binding proteins known as septins. These have been divided into four different subgroups on the basis of sequence similarity. The differences between the subgroups are believed to control their correct assembly into heterofilaments which have specific roles in membrane remodelling events. Many different combinations of the 13 proteins are theoretically possible and it is therefore important to understand the structural basis of specific filament assembly. However, three-dimensional structures are currently available for only three of the four subgroups. In the present study we describe the crystal structure of a construct of human SEPT3 which belongs to the outstanding subgroup. This construct (SEPT3-GC), which includes the GTP-binding and C-terminal domains, purifies as a nucleotide-free monomer, allowing for its characterization in terms of GTP-binding and hydrolysis. In the crystal structure, SEPT3-GC forms foreshortened filaments which employ the same NC and G interfaces observed in the heterotrimeric complex of human septins 2, 6 and 7, reinforcing the notion of ‘promiscuous’ interactions described previously. In the present study we describe these two interfaces and relate the structure to its tendency to form monomers and its efficiency in the hydrolysis of GTP. The relevance of these results is emphasized by the fact that septins from the SEPT3 subgroup may be important determinants of polymerization by occupying the terminal position in octameric units which themselves form the building blocks of at least some heterofilaments.

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High resolution spot scan imaging of frozen, hydrated actin bundles with 400kV electrons

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122964 ◽

1992 ◽

Vol 50 (1) ◽

pp. 512-513

Author(s):

J. Jakana ◽

M.F. Schmid ◽

P. Matsudaira ◽

W. Chiu

Keyword(s):

High Resolution ◽

Binding Proteins ◽

Actin Binding ◽

Eukaryotic Cells ◽

Dimensional Structure ◽

Structural Basis ◽

Actin Bundle ◽

Actin Bundles ◽

3 Dimensional ◽

Macromolecular Assembly

Actin is a protein found in all eukaryotic cells. In its polymerized form, the cells use it for motility, cytokinesis and for cytoskeletal support. An example of this latter class is the actin bundle in the acrosomal process from the Limulus sperm. The different functions actin performs seem to arise from its interaction with the actin binding proteins. A 3-dimensional structure of this macromolecular assembly is essential to provide a structural basis for understanding this interaction in relationship to its development and functions.

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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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Crystal Structures of Fragments D and Double-D from Fibrinogen and Fibrin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615842 ◽

1999 ◽

Vol 82 (08) ◽

pp. 271-276 ◽

Cited By ~ 8

Author(s):

Glen Spraggon ◽

Stephen Everse ◽

Russell Doolittle

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Crystal Structures ◽

Structure And Function ◽

X Ray ◽

Long Period ◽

And Function

IntroductionAfter a long period of anticipation,1 the last two years have witnessed the first high-resolution x-ray structures of fragments from fibrinogen and fibrin.2-7 The results confirmed many aspects of fibrinogen structure and function that had previously been inferred from electron microscopy and biochemistry and revealed some unexpected features. Several matters have remained stubbornly unsettled, however, and much more work remains to be done. Here, we review several of the most significant findings that have accompanied the new x-ray structures and discuss some of the problems of the fibrinogen-fibrin conversion that remain unresolved. * Abbreviations: GPR—Gly-Pro-Arg-derivatives; GPRPam—Gly-Pro-Arg-Pro-amide; GHRPam—Gly-His-Arg-Pro-amide

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The crystal structures of the β1and β2polymorphs of mono-unsaturated triacylglycerols and cocoa butter determined from high resolution powder diffraction data

Zeitschrift für Kristallographie Supplements ◽

10.1524/zksu.2007.2007.suppl_26.599 ◽

2007 ◽

Vol 2007 (suppl_26) ◽

pp. 599-604 ◽

Cited By ~ 2

Author(s):

J. B. van Mechelen ◽

R. Peschar ◽

H. Schenk

Keyword(s):

High Resolution ◽

Cocoa Butter ◽

Crystal Structures ◽

Powder Diffraction ◽

Diffraction Data ◽

Powder Diffraction Data

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Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

Nature Communications ◽

10.1038/s41467-021-24421-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhiwen Liu ◽

Fanglong Zhao ◽

Boyang Zhao ◽

Jie Yang ◽

Joseph Ferrara ◽

...

Keyword(s):

High Resolution ◽

Organic Synthesis ◽

Indole Alkaloids ◽

Site Directed Mutagenesis ◽

Structural Basis ◽

Directed Mutagenesis ◽

Computational Studies ◽

X Ray ◽

Evolutionary Branch ◽

Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

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