An increasing number of SH3 domain–ligand interactions continue to be described that involve the conserved peptide-binding surface of SH3, but structurally deviate substantially from canonical ...docking of consensus motif-containing SH3 ligands. Indeed, it appears that that the relative frequency and importance of these types of interactions may have been underestimated. Instead of atypical, we propose referring to such peptides as type I or II (depending on the binding orientation) non-consensus ligands. Here we discuss the structural basis of non-consensus SH3 ligand binding and the dominant role of the SH3 domain specificity zone in selective target recognition, and review some of the best-characterized examples of such interactions.
Unidirectional coherence transfer is highly efficient in intrinsically disordered proteins (IDPs). Their elevated ps-ns timescale dynamics ensures long transverse (T
2
) relaxation times allowing ...sophisticated coherence transfer pathway selection in comparison to folded proteins.
1
H
α
-detection ensures non-susceptibility to chemical exchange with the solvent and enables chemical shift assignment of consecutive proline residues, typically abundant in IDPs. However, many IDPs undergo a disorder-to-order transition upon interaction with their target protein, which leads to the loss of the favorable relaxation properties. Long coherence transfer routes now result in prohibitively large decrease in sensitivity. We introduce a novel 4D
1
H
α
-detected experiment HACANCOi, together with its 3D implementation, which warrant high sensitivity for the assignment of proline-rich regions in IDPs in complex with a globular protein. The experiment correlates
1
H
α
i
,
13
C
α
i
,
15
N
i
and
13
C
i
′
spins by transferring the magnetization concomitantly from
13
C
α
i
to
15
N
i
and
13
C
i
′
. The B1 domain of protein G (GB1), and the enteropathogenic
E.
coli
EspF in complex with human SNX9 SH3, serve as model systems to demonstrate the attainable sensitivity and successful sequential assignment.
Physical activity is essential in weight management, improves overall health, and mitigates obesity-related risk markers. Besides inducing changes in systemic metabolism, habitual exercise may ...improve gut's microbial diversity and increase the abundance of beneficial taxa in a correlated fashion. Since there is a lack of integrative omics studies on exercise and overweight populations, we studied the metabolomes and gut microbiota associated with programmed exercise in obese individuals. We measured the serum and fecal metabolites of 17 adult women with overweight during a 6-week endurance exercise program. Further, we integrated the exercise-responsive metabolites with variations in the gut microbiome and cardiorespiratory parameters. We found clear correlation with several serum and fecal metabolites, and metabolic pathways, during the exercise period in comparison to the control period, indicating increased lipid oxidation and oxidative stress. Especially, exercise caused co-occurring increase in levels of serum lyso-phosphatidylcholine moieties and fecal glycerophosphocholine. This signature was associated with several microbial metagenome pathways and the abundance of Akkermansia. The study demonstrates that, in the absence of body composition changes, aerobic exercise can induce metabolic shifts that provide substrates for beneficial gut microbiota in overweight individuals.
The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. ...A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.
Antibiotic resistance is a growing problem and a global threat for modern healthcare. New approaches complementing the traditional antibiotic drugs are urgently needed to secure the ability to treat ...bacterial infections also in the future. Among the promising alternatives are bacteriolytic enzymes, such as the cell wall degrading peptidoglycan hydrolases.
Staphylococcus aureus
LytM, a Zn
2+
-dependent glycyl-glycine endopeptidase of the M23 family, is one of the peptidoglycan hydrolases. It has a specificity towards staphylococcal peptidoglycan, making it an interesting target for antimicrobial studies. LytM hydrolyses the cell wall of
S. aureus
, a common pathogen with multi-resistant strains that are difficult to treat, such as the methicillin-resistant
S. aureus
, MRSA. Here we report the
1
H,
15
N and
13
C chemical shift assignments of
S. aureus
LytM N-terminal domain and linker region, residues 26–184. These resonance assignments can provide the basis for further studies such as elucidation of structure and interactions.
Resonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if ...repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three H
α
-detected 4D experiments with C
α
as an additional dimension. In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D H
α
-start, H
N
-detect experiments which have two N
H
dimensions to enhance peak dispersion in a sequential walk through C′, N
H
and H
N
, and provide more accurate N
H
/H
N
chemical shifts than those that can be obtained from a crowded
1
H,
15
N-HSQC spectrum. Application of these 4D experiments is demonstrated using BilRI (165 aa), an outer-membrane intrinsically disordered protein from the opportunistic oral pathogen
Aggregatibacter actinomycetemcomitans
. BilRI amino acid sequence encompasses three very similar repeats with a 13-residue identical stretch in two of them.
Cyanobacteria produce a wide variety of cyclic peptides, including the widespread hepatotoxins microcystins and nodularins. Another class of peptides, cyclic glycosylated lipopeptides called ...hassallidins, show antifungal activity. Previously, two hassallidins (A and B) were reported from an epilithic cyanobacterium Hassallia sp. and found to be active against opportunistic human pathogenic fungi. Bioinformatic analysis of the Anabaena sp. 90 genome identified a 59-kb cryptic inactive nonribosomal peptide synthetase gene cluster proposed to be responsible for hassallidin biosynthesis. Here we describe the hassallidin biosynthetic pathway from Anabaena sp. SYKE748A, as well as the large chemical variation and common occurrence of hassallidins in filamentous cyanobacteria. Analysis demonstrated that 20 strains of the genus Anabaena carry hassallidin synthetase genes and produce a multitude of hassallidin variants that exhibit activity against Candida albicans . The compounds discovered here were distinct from previously reported hassallidins A and B. The IC ₅₀ of hassallidin D was 0.29–1.0 µM against Candida strains. A large variation in amino acids, sugars, their degree of acetylation, and fatty acid side chain length was detected. In addition, hassallidins were detected in other cyanobacteria including Aphanizomenon , Cylindrospermopsis raciborskii , Nostoc , and Tolypothrix . These compounds may protect some of the most important bloom-forming and globally distributed cyanobacteria against attacks by parasitic fungi.
S. aureus
resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called “last-resort” drugs. ...Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant
S. aureus
is high. M23B family peptidoglycan hydrolases, enzymes that can kill
S. aureus
by cleaving glycine-glycine peptide bonds in
S. aureus
cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. We report here the near complete
1
H/
13
C/
15
N resonance assignment of the catalytic domain of LytM, residues 185–316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall.
We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is ...highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the overall structure common to the lysostaphin family. Purified LytU lyses S. aureus cells and cleaves pentaglycine, a reaction conveniently monitored by NMR spectroscopy. Substituting the cofactor zinc ion with a copper or cobalt ion remarkably increases the rate of pentaglycine cleavage. NMR and isothermal titration calorimetry further reveal that, uniquely for its family, LytU is able to bind a second zinc ion which is coordinated by catalytic histidines and is therefore inhibitory. The pH-dependence and high affinity of binding carry further physiological implications.
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