Structural Flexibility of Peripheral Loops and Extended C-Term Domain of Short Length Substrate Binding Protein from Rhodothermus marinus

Substrate binding proteins (SBP) bind to specific ligands in the periplasmic region and bind to membrane proteins to participate in transport or signal transduction. Typical SBPs consist of two α/β domains and recognize the substrate by hinge motion between two domains. Conversely, short length Rhodothermus marinus SBP (named as RmSBP) exists around the methyl-accepting chemotaxis protein. We previously determined the crystal structure of RmSBP consisting of a single α/β domain, but the substrate recognition mechanism is still unclear. To better understand the short length RmSBP, we performed comparative structure analysis, computational substrate docking, and X-ray crystallographic study. RmSBP shares a high level of similarity in α/β domain with other SBP proteins, but it has a distinct topology in the C-term region. The substrate binding model suggested that conformational change in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility of the β1-α2 loop, β5-β6 loop, and extended C-term domain based on the electron density map and temperature B-factor analysis. These results provide information that will further the understanding of the function of the short length SBP.

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Structural Flexibility of Peripheral Loops and Extended C-terminal Domain of Short Length Substrate Binding Protein from Rhodothermus marinus

The Protein Journal ◽

10.1007/s10930-021-09970-z ◽

2021 ◽

Author(s):

Ji-Eun Bae ◽

In Jung Kim ◽

Yongbin Xu ◽

Ki Hyun Nam

Keyword(s):

Binding Protein ◽

Substrate Binding ◽

Short Length ◽

Structural Flexibility ◽

Rhodothermus Marinus ◽

Terminal Domain ◽

Substrate Binding Protein

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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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Thermal Analysis for Geologic Codisposal of SNF Packages

10th International Conference on Nuclear Engineering, Volume 4 ◽

10.1115/icone10-22390 ◽

2002 ◽

Author(s):

Si Y. Lee

Keyword(s):

Thermal Analysis ◽

Nuclear Fuel ◽

Spent Nuclear Fuel ◽

Peripheral Region ◽

Waste Form ◽

Temperature History ◽

High Level Waste ◽

Geologic Repository ◽

Geologic Disposal ◽

High Level

The engineering viability of disposal of aluminum-clad, aluminum-based spent nuclear fuel (Al-SNF) in a geologic repository requires a thermal analysis to provide the temperature history of the waste form. Calculated temperatures are used to demonstrate compliance with criteria for waste acceptance into the geologic disposal system and as input to assess the chemical and physical behavior of the waste form within the Waste Package (WP). The leading codisposal WP design proposes that a central DOE Al-SNF canister be surrounded by five Defense Waste Process Facility (DWPF) glass log canisters, that is, High-level Waste Glass Logs (HWGL’s), and placed into a WP in a geologic disposal system. A DOE SNF canister having about 0.4318m diameter is placed along the central horizontal axis of the WP. The five HWGL’s will be located around the peripheral region of the DOE SNF canister within the cylindrical WP container. The codisposal WP will be laid down horizontally in a drift repository. In this situation, two waste form options for Al-SNF disposition are considered using the codisposal WP design configurations. They are the direct Al-SNF form and the melt-dilute ingot. In the present work, the reference geologic and design conditions are assumed for the analysis even though the detailed package design is continuously evolved. This paper primarily dealt with the thermal performance internal to the codisposal WP for the qualification study of the WP containing Al-SNF. Thermal analysis methodology and decay heat source terms have been developed to calculate peak temperatures and temperature profiles of Al-SNF package in the DOE spent nuclear fuel canister within the geologic codisposal WP.

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Structural Insights into the Multispecific Recognition of Dipeptides of Deep-Sea Gram-Negative Bacterium Pseudoalteromonas sp. Strain SM9913

Journal of Bacteriology ◽

10.1128/jb.02600-14 ◽

2015 ◽

Vol 197 (6) ◽

pp. 1125-1134 ◽

Cited By ~ 5

Author(s):

Chun-Yang Li ◽

Xiu-Lan Chen ◽

Qi-Long Qin ◽

Peng Wang ◽

Wei-Xin Zhang ◽

...

Keyword(s):

Nitrogen Cycling ◽

Marine Bacteria ◽

Deep Sea ◽

Substrate Binding ◽

Substrate Recognition ◽

Recognition Mechanism ◽

Gram Negative ◽

Content Type ◽

Peptide Uptake ◽

Structural Insights

ABSTRACTPeptide uptake is important for nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide binding protein of dipeptide permease (Dpp; an important peptide transporter in bacteria) and exclusively controls the substrate specificity of Dpp. Here, the substrate binding specificity of deep-seaPseudoalteromonassp. strain SM9913 DppA (PsDppA) was analyzed for 25 different dipeptides with various properties by using isothermal titration calorimetry measurements.PsDppA showed binding affinities for 8 dipeptides. To explain the multispecific substrate recognition mechanism ofPsDppA, we solved the crystal structures of unligandedPsDppA and ofPsDppA in complex with 4 different types of dipeptides (Ala-Phe, Met-Leu, Gly-Glu, and Val-Thr).PsDppA alternates between an “open” and a “closed” form during substrate binding. Structural analyses of the 4PsDppA-substrate complexes combined with mutational assays indicate thatPsDppA binds to different substrates through a precise mechanism: dipeptides are bound mainly by the interactions between their backbones andPsDppA, in particular by anchoring their N and C termini through ion-pair interactions; hydrophobic interactions are important in binding hydrophobic dipeptides; and Lys457 is necessary for the binding of dipeptides with a C-terminal glutamic acid or glutamine. Additionally, sequence alignment suggests that the substrate recognition mechanism ofPsDppA may be common in Gram-negative bacteria. All together, our results provide structural insights into the multispecific substrate recognition mechanism of marine Gram-negative bacterial DppA, which provides a better understanding of the mechanisms of marine bacterial peptide uptake.IMPORTANCEPeptide uptake plays a significant role in nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria recognize and absorb peptides is still unclear. This study analyzed the substrate binding specificity of deep-seaPseudoalteromonassp. strain SM9913 DppA (PsDppA; the dipeptide-binding protein of dipeptide permease) and solved the crystal structures of unligandedPsDppA andPsDppA in complex with 4 different types of dipeptides. The multispecific recognition mechanism ofPsDppA for dipeptides is explained based on structural and mutational analyses. We also find that the substrate-binding mechanism ofPsDppA may be common in Gram-negative bacteria. This study sheds light on marine Gram-negative bacterial peptide uptake and marine nitrogen cycling.

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