I04-Macromolecular Crystallography
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Open Access
Abstract: We have recently demonstrated a novel anaerobic NADH-dependent haem breakdown reaction, which is carried out by a range of haemoproteins. The Yersinia enterocolitica protein, HemS, is the focus of further research presented in the current paper. Using conventional experimental methods, bioinformatics, and energy landscape theory (ELT), we provide new insight into the mechanism of the novel breakdown process. Of particular interest is the behavior of a double phenylalanine gate, which opens and closes according to the relative situations of haem and NADH within the protein pocket. This behavior suggests that the double phe-gate fulfills a regulatory role within the pocket, controlling the access of NADH to haem. Additionally, stopped-flow spectroscopy results provide kinetic comparisons between the wild-type and the selected mutants. We also present a fully resolved crystal structure for the F104AF199A HemS monomer, including its extensive loop, the first such structure to be completely resolved for HemS or any of its close homologues. The energy landscapes approach provided key information regarding the gating strategy employed by HemS, compensating for current limitations with conventional biophysical and molecular dynamics approaches. We propose that ELT become more widely used in the field, particularly in the investigation of the dynamics and interactions of weak-binding ligands, and for gating features, within protein cavities.
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Feb 2025
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I24-Microfocus Macromolecular Crystallography
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Maria Elena
Laugieri
,
Immacolata
Speciale
,
Ana
Gimeno
,
Sicheng
Lin
,
Brock W.
Byers
,
Ana
Poveda
,
Reyes
Núñez‐franco
,
Idoia
Iturrioz
,
María J.
Moure
,
Gonzalo
Jiménez-Osés
,
Irene
Russo‐krauss
,
Anna
Notaro
,
James L.
Van Etten
,
Todd L.
Lowary
,
Jesús
Jimenez-Barbero
,
Cristina
De Castro
,
Michela
Tonetti
,
Adriana L.
Rojas
Diamond Proposal Number(s):
[20113]
Open Access
Abstract: Protein A075L is a β-xylosyltransferase that participates in producing the core of the N-glycans found in VP54, the major viral capsid protein of Paramecium bursaria chlorella virus-1 (PBCV-1). In this study, we present an X-ray crystallographic analysis of the apo form of A075L, along with its complexes with the sugar donor and with a trisaccharide acceptor. The protein structure shows a typical GT-B folding, with two Rossmann-like fold domains, in which the acceptor substrate binds to the N-terminal region, and the nucleotide-sugar donor binds to the C-terminal region. We propose that the catalytic mechanism follows a direct displacement SN2-like reaction, where Asp73 serves as a catalytic base that deprotonates the incoming nucleophile of the acceptor, facilitating direct displacement of the UDP with the inversion of the anomeric configuration of the acceptor without metal ion dependence, while the interactions with side chains of Arg158 and Arg208 stabilize the developing negative charge. Using isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, and molecular dynamics simulations, the catalytic activity and specificity of this enzyme have been unraveled.
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Dec 2024
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I04-Macromolecular Crystallography
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Abstract: In Escherichia coli AcrB is a major multidrug exporter, which confers the bacterium resistance to many antibiotics with diverse structural and chemical proprieties. Studies have identified three possible tunnels (or channels) within AcrB that different substrates use before reaching the distal pocket, from which they are subsequently extruded. Recently, we reported that mutations in the AcrB gate loop may affect the conformational change kinetics involved in substrate export rather than directly affecting molecular interactions with this loop, and we highlighted the distinct export tunnel preferences between erythromycin and doxorubicin. To further understand the gate loop's role in AcrB's export activity and the rationale behind substrate preferences among the three possible export tunnels, namely tunnel-1, -2, and -3, we investigated the structural and functional effects of several single and multiple mutations in the gate loop of AcrB. Our findings indicate that all three tunnels are energetically favorable for the substrates studied, with the majority forming more hydrogen bonds in any tunnel compared to the distal pocket. Moreover, our experimental and computational data revealed that some substrates with high molecular similarity exhibited different export tunnel preferences, as strongly suggested by their MIC values. To explain this unexpected outcome, we propose a generalized explanation that the conformational change kinetics in AcrB is substrate-dependent.
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Dec 2024
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[25108, 32728]
Open Access
Abstract: Decades of research describe myriad redox enzymes that contain cofactors arranged in tightly packed chains facilitating rapid and controlled intra-protein electron transfer. Many such enzymes participate in extracellular electron transfer (EET), a process which allows microorganisms to conserve energy in anoxic environments by exploiting mineral oxides and other extracellular substrates as terminal electron acceptors. In this work, we describe the properties of the triheme cytochrome PgcA from Geobacter sulfurreducens. PgcA has been shown to play an important role in EET but is unusual in containing three CXXCH heme binding motifs that are separated by repeated (PT)x motifs, suggested to enhance binding to mineral surfaces. Using a combination of structural, electrochemical, and biophysical techniques, we experimentally demonstrate that PgcA adopts numerous conformations stretching as far as 180 Å between the ends of domains I and III, without a tightly packed cofactor chain. Furthermore, we demonstrate a distinct role for its domain III as a mineral reductase that is recharged by domains I and II. These findings show PgcA to be the first of a new class of electron transfer proteins, with redox centers separated by some nanometers but tethered together by flexible linkers, facilitating electron transfer through a tethered diffusion mechanism rather than a fixed, closely packed electron transfer chain.
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Nov 2024
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I03-Macromolecular Crystallography
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Open Access
Abstract: Crystallography at low resolution must determine the atomic model from less experimental observations, which is challenging in the absence of a model. In addition, model bias is more severe when independent experimental data are scarce. Our methods solve the phase problem by combining the location of accurate model fragments using Phaser with density modification and interpretation of the resulting maps using SHELXE. From a partial, correct structure, the density modification process and the stereochemical constraints draw the rest of the structure, validating the result. This same principle is now exploited at low resolution. Coiled coils are important, ubiquitous structures but notoriously difficult to phase and to predict. Both correct solutions and incorrect ones are poorly discriminated by the crystallographic figures of merit as long as helices are correctly oriented. We incorporate coiled-coil verification, designed to set up competing, incompatible structural hypotheses to probe both the results and establish the power of the data to discriminate them. Efficiency of coiled-coil phasing and validation in test cases from 3 to 4 Å is demonstrated in ARCIMBOLDO_LITE, placing single helices, and in ARCIMBOLDO_SHREDDER, with fragments derived from AlphaFold models. SHELXE tracing at low resolution has been enhanced, maintaining its local character but extending the environment assessment. For non-helical structures, verification is demonstrated in the fragment location process. Its use is exemplified with the solution of the VSR1 structure at 3.5 Å, depending on LLG optimization and the emergence of new features in the electron density. Relying on verification, we have extended the use of the ARCIMBOLDO software to low resolution.
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Sep 2024
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B21-High Throughput SAXS
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Gefei
Chen
,
Yu
Wang
,
Zihan
Zheng
,
Wangshu
Jiang
,
Axel
Leppert
,
Xueying
Zhong
,
Anna
Belorusova
,
Gregg
Siegal
,
Caroline
Jegerschold
,
Philip J. B.
Koeck
,
Axel
Abelein
,
Hans
Hebert
,
Stefan D.
Knight
,
Jan
Johansson
Open Access
Abstract: Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.
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Jul 2024
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[20113]
Open Access
Abstract: Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin-polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of FAM21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from FAM21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between FAM21 and retromer occurring near the Parkinson's disease-linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with FAM21 in vitro.
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May 2024
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B21-High Throughput SAXS
I23-Long wavelength MX
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Carlos
Vela-Rodríguez
,
Chunsong
Yang
,
Heli I.
Alanen
,
Rebeka
Eki
,
Tarek A.
Abbas
,
Mirko M.
Maksimainen
,
Tuomo
Glumoff
,
Ramona
Duman
,
Armin
Wagner
,
Bryce M.
Paschal
,
Lari
Lehtio
Diamond Proposal Number(s):
[26794]
Open Access
Abstract: Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.
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Apr 2024
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme.
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Apr 2024
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Emily M.
Cross
,
Nasim
Akbari
,
Hanieh
Ghassabian
,
Mikayla
Hoad
,
Silvia
Pavan
,
Daryl
Ariawan
,
Camilla M.
Donnelly
,
Enrico
Lavezzo
,
Gayle F.
Petersen
,
Jade K.
Forwood
,
Gualtiero
Alvisi
Open Access
Abstract: Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPβ1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, whilst bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα-cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) Large Tumor Antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full-length proteins. While several LTAs possess bipartite cNLSs, Merkel Cell (MC) PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site.
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Dec 2023
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