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Disordered scaffold proteins provide multivalent landing pads that, via a series of embedded Short Linear Motifs (SLiMs), bring together the components of a complex to orchestrate precise spatial and temporal regulation of cellular processes. One such protein is AKAP5 (previously AKAP79), which contains SLiMs that anchor PKA and Calcineurin, and recruit substrate (the TRPV1 receptor). Calcineurin is anchored to AKAP5 by a well-characterised PxIxIT SLiM. Here we show, using a combination of biochemical and biophysical approaches, that the Calcineurin PxIxIT-binding groove also recognises several hitherto unknown lower-affinity SLiMs in addition to the PxIxIT motif. We demonstrate that the assembly is in reality a complex system with conserved SLiMs spanning a wide affinity range. The capture is analogous to that seen for many DNA-binding proteins that have a weak non-specific affinity for DNA outside the canonical binding site, but different in that it involves (i) two proteins, and (ii) hydrophobic rather than electrostatic interactions. It is also compatible with the requirement for both stable anchoring of the enzyme and responsive downstream signalling. We conclude that the AKAP5 C-terminus is enriched in lower-affinity/mini-SLiMs that, together with the canonical SLiM, maintain a structurally disordered but tightly regulated signalosome., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: Acute myelogenous leukemia (AML) largely affects older adults. Few interventions have sought to improve functional status and health-related quality of life (HRQL) during treatment. The objective of this study is to examine the feasibility of an exercise intervention among older adults with AML undergoing induction chemotherapy., Materials and Methods: Pilot study of adults ≥50 years of age hospitalized for AML chemotherapy. The four week exercise intervention included stretching, walking, and strength exercises. Feasibility measures included recruitment, retention, number of exercise sessions completed, and barriers to participation. Physical function, HRQL, depression, and distress were assessed at baseline (week 1), upon completion of intervention (week 5), and during follow-up (weeks 9-13). Exploratory analyses used repeated measures ANCOVA to model changes over time., Results: Among 55 eligible inpatients, 24 enrolled (43.6%). Mean age was 65.1 years (SD 7.8). 87.5% of participants completed baseline measures; 70.8% attended ≥1 exercise sessions, and 50.0% completed post-intervention assessment. Among baseline characteristics, only higher physical performance was associated with greater number of exercise sessions attended (p=0.001). Post intervention, HRQL and depressive symptoms improved (p <0.05)., Conclusions: Recruitment to an exercise intervention was feasible. Exercise shows promise to maintain physical function and enhance HRQL. Strategies to enhance adherence to exercise are needed to maximize benefit.

The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1-DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box-tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. (15)N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl)methyl methanethiosulfonate] placed at three locations in the tail confirm our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements of B domain residues, suggesting the existence of a "sandwich"-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein-protein interactions or posttranslational modifications might regulate the function of the protein at particular sites, or at particular stages in the cell cycle., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

H1 and HMGB1 bind to linker DNA in chromatin, in the vicinity of the nucleosome dyad. They appear to have opposing effects on the nucleosome, H1 stabilising it by "sealing" two turns of DNA around the octamer, and HMGB1 destabilising it, probably by bending the adjacent DNA. Their presence in chromatin might be mutually exclusive. Displacement/replacement of one by the other as a result of their highly dynamic binding in vivo might, in principle, involve interactions between them. Chemical cross-linking and gel-filtration show that a 1:1 linker histone/HMGB1 complex is formed, which persists at physiological ionic strength, and that complex formation requires the acidic tail of HMGB1. NMR spectroscopy shows that the linker histone binds, predominantly through its basic C-terminal domain, to the acidic tail of HMGB1, thereby disrupting the interaction of the tail with the DNA-binding faces of the HMG boxes. A potential consequence of this interaction is enhanced DNA binding by HMGB1, and concomitantly lowered affinity of H1 for DNA. In a chromatin context, this might facilitate displacement of H1 by HMGB1.

The numbers of frail older persons using emergency departments are already considerable and will continue to increase over time. There are a number of issues related to the assessment and care of older patients that are significantly different to other patient groups. The traditional emergency department (ED) model focusing on rapid triage, treatment and throughput does not meet the needs of many older patients, who have complex presentations, and require comprehensive assessment and referral. In response to this, there are already a number of appropriate and innovative approaches to the care of the older patient demonstrated in the literature. Nurses have a crucial role in contributing to these approaches and in raising the profile of quality care of the older person. Some specific areas that ED nurses can focus on include a more comprehensive approach to assessment and discharge planning, improved communication with the patient and their personal carers, attention to basic nursing care, and making the physical environment safer and less stressful for the older patient. While developing collaborations with their aged care nursing colleagues is important, emergency nurses need to view care of the older person as a central part of their own core business.

The mechanism underlying negative regulation of HMGB1-DNA interaction by the acidic C-terminal tail is ill defined. To address this issue, we have devised a novel NMR chemical-shift perturbation mapping strategy to elucidate interactions between the tail, which consists solely of aspartic acid and glutamic acid residues, and the two well characterized HMG-box DNA-binding domains. A series of HMGB1 tail-truncation mutants differing from each other by five residues was generated. Chemical-shift perturbation mapping using these mutants shows that tails of different lengths bind with different affinities. Nevertheless, the truncated tails bind along the same path on the HMG boxes as the full-length tail, differences in length being manifested in differences in the "reach". The tail makes extensive contacts with the DNA-binding surfaces of both HMG boxes, thus explaining the basis of negative regulation of HMGB1-DNA interaction by the tail.

The high-mobility group protein HMGB1 contains two tandem DNA-binding HMG box domains, A and B, linked by a short flexible linker that allows the two domains to behave independently in the free protein. There is no structural information on how the linked domains and linker behave when bound to DNA, mainly due to the lack of any DNA-sequence preference of HMGB1. We report the structure determination, by NMR spectroscopy, of a well-defined complex of two tandem HMG boxes bound to a 16 bp oligonucleotide. The protein is an engineered version of the AB di-domain of HMGB1, in which the A box has been replaced by the HMG box of the sequence-specific transcription factor SRY, to give SRY.B. In the SRY.B/DNA complex, both HMG boxes bind in the minor groove and contribute to the overall DNA bending by intercalation of bulky hydrophobic residues between base-pairs; the bends reinforce each other, and the basic linker lies partly in the minor groove. As well as being the first structure of an HMG-box di-domain bound to DNA, this provides the first structure of the B domain of HMGB1 bound to DNA.

Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA ( approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site ("site II") on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two "missing" site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.

The Saccharomyces cerevisiae homologue of the linker histone H1, Hho1p, has two domains that are similar in sequence to the globular domain of H1 (and variants such as H5). It is an open question whether both domains are functional and whether they play similar structural roles. Preliminary structural studies showed that the two isolated domains, GI and GII, differ significantly in stability. In 10 mM sodium phosphate (pH 7), the GI domain, like the globular domains of H1 and H5, GH1 and GH5, was stably folded, whereas GII was largely unstructured. However, at high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate), which might mimic the charge-screening effects of DNA phosphate groups, GII was folded. In view of the potential significance of these observations in relation to the role of Hho1p, we have now determined the structures of its GI and GII domains by NMR spectroscopy under conditions in which GII (like GI) is folded. The backbone r.m.s.d. over the ordered residues is 0.43 A for GI and 0.97 A for GII. Both structures show the "winged-helix" fold typical of GH1 and GH5 and are very similar to each other, with an r.m.s.d. over the structured regions of 1.3 A, although there are distinct differences. The potential for GII to adopt a structure similar to that of GI when Hho1p is bound to chromatin in vivo suggests that both globular domains might be functional. Whether Hho1p performs a structural role by bridging two nucleosomes remains to be determined.

Previous peptide dissection and kinetic experiments have indicated that in vitro folding of ubiquitin may proceed via transient species in which native-like structure has been acquired in the first 45 residues. A peptide fragment, UQ(1-51), encompassing residues 1 to 51 of ubiquitin was produced in order to test whether this portion has propensity for independent self-assembly. Surprisingly, the construct formed a folded symmetrical dimer that was stabilised by 0.8 M sodium sulphate at 298 K (the S state). The solution structure of the UQ(1-51) dimer was determined by multinuclear NMR spectroscopy. Each subunit of UQ(1-51) consists of an N-terminal beta-hairpin followed by an alpha-helix and a final beta-strand, with orientations similar to intact ubiquitin. The dimer is formed by the third beta-strand of one subunit interleaving between the hairpin and third strand of the other to give a six-stranded beta-sheet, with the two alpha-helices sitting on top. The helix-helix and strand portions of the dimer interface also mimic related features in the structure of ubiquitin. The structural specificity of the UQ(1-51) peptide is tuneable: as the concentration of sodium sulphate is decreased, near-native alternative conformations are populated in slow chemical exchange. Magnetization transfer experiments were performed to characterize the various species present in 0.35 M sodium sulphate, namely the S state and two minor forms. Chemical shift differences suggest that one minor form is very similar to the S state, while the other experiences a significant conformational change in the third strand. A segmental rearrangement of the third strand in one subunit of the S state would render the dimer asymmetric, accounting for most of our results. Similar small-scale transitions in proteins are often invoked to explain solvent exchange at backbone amide proton sites that have an intermediate level of protection., (Copyright 2001 Academic Press.)

Mutants of chymotrypsin inhibitor protein 2 have previously been studied in which 4 or 10 glutamine residues were inserted into the inhibitory loop of the protein between residues 59 and 60, as potential models for the behaviour of glutamine tracts in proteins associated with polyglutamine-expansion neurodegenarative diseases. These mutants form very stable monomers, dimers and trimers. Although the cause of oligomerisation was found to be domain-swapping, it was thought that the glutamine insertions might nevertheless show evidence of weak interglutamine interactions in solution that could mimic those occurring in disease-associated proteins. In the present NMR study, we used steady-state (15)N[(1)H] NOE measurements and chemical shift comparisons to characterise the motional properties of the inserted glutamines in these CI2 mutants. We found the glutamines to be highly mobile, with no evidence of interactions amongst them in either monomers or dimers., (Copyright 2001 Academic Press.)

Reductive acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed specifically by its partner pyruvate decarboxylase (E1p), and no productive interaction occurs with the analogous 2-oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase complex. Residues in the lipoyl-lysine beta-turn region of the unlipoylated E2plip domain (E2plip(apo)) undergo significant changes in both chemical shift and transverse relaxation time (T(2)) in the presence of E1p but not E1o. Residue Gly11, in a prominent surface loop between beta-strands 1 and 2 in the E2plip domain, was also observed to undergo a significant change in chemical shift. Addition of pyruvate to the mixture of E2plip(apo) and E1p caused larger changes in chemical shift and the appearance of multiple cross-peaks for certain residues, suggesting that the domain was experiencing more than one type of interaction. Residues in both beta-strands 4 and 5, together with those in the prominent surface loop and the following beta-strand 2, appeared to be interacting with E1p, as did a small patch of residues centred around Glu31. The values of T(2) across the polypeptide chain backbone were also lower than in the presence of E1p alone, suggesting that E2plip(apo) binds more tightly after the addition of pyruvate. The lipoylated domain (E2plip(holo)) also exhibited significant changes in chemical shift and decreases in the overall T(2) relaxation times in the presence of E1p, the residues principally affected being restricted to the half of the domain that contains the lipoyl-lysine (Lys41) residue. In addition, small chemical shift changes and a general drop in T(2) times in the presence of E1o were observed, indicating that E2plip(holo) can interact, weakly but non-productively, with E1o. It is evident that recognition of the protein domain is the ultimate determinant of whether reductive acetylation of the lipoyl group occurs, and that this is ensured by a mosaic of interactions with the Elp., (Copyright 2001 Academic Press.)