The AlignMe web server is dedicated to accurately aligning sequences of membrane proteins, a particularly challenging task due to the strong evolutionary divergence and the low compositional complexity of hydrophobic membrane-spanning proteins. AlignMe can create pairwise alignments of either two primary amino acid sequences or two hydropathy profiles. The web server for AlignMe has been continuously available for >10 years, supporting 1000s of users per year. Recent improvements include anchoring, multiple submissions, and structure visualization. Anchoring is the ability to constrain a position in an alignment, which allows expert information about related residues in proteins to be incorporated into an alignment without manual modification. The original web interface to the server limited the user to one alignment per submission, hindering larger scale studies. Now, batches of alignments can be initiated with a single submission. Finally, to provide structural context for the relationship between proteins, sequence similarity can now be mapped onto one or more structures (or structural models) of the proteins being aligned, by links to MutationExplorer, a web-based visualization tool. Together with a refreshed user interface, these features further enhance an important resource in the membrane protein community. The AlignMe web server is freely available at https://www.bioinfo.mpg.de/AlignMe/.

The alignment of primary sequences is a fundamental step in the analysis of protein structure, function, and evolution, and in the generation of homology-based models. Integral membrane proteins pose a significant challenge for such sequence alignment approaches, because their evolutionary relationships can be very remote, and because a high content of hydrophobic amino acids reduces their complexity. Frequently, biochemical or biophysical data is available that informs the optimum alignment, for example, indicating specific positions that share common functional or structural roles. Currently, if those positions are not correctly matched by a standard pairwise sequence alignment procedure, the incorporation of such information into the alignment is typically addressed in an ad hoc manner, with manual adjustments. However, such modifications are problematic because they reduce the robustness and reproducibility of the aligned regions either side of the newly matched positions. Previous studies have introduced restraints as a means to impose the matching of positions during sequence alignments, originally in the context of genome assembly. Here we introduce position restraints, or “anchors” as a feature in our alignment tool AlignMe, providing an aid to pairwise global sequence alignment of alpha-helical membrane proteins. Applying this approach to realistic scenarios involving distantly-related and low complexity sequences, we illustrate how the addition of anchors can be used to modify alignments, while still maintaining the reproducibility and rigor of the rest of the alignment. Anchored alignments can be generated using the online version of AlignMe available at www.bioinfo.mpg.de/AlignMe/.

Multivalent interactions of intrinsically disordered segments of proteins are essential in many cellular functions, but challenges in recombinant protein production has limited understanding of how the complexes assemble. One example is the interaction between Angiomotin-like 1 (AMOTL1), Yesassociated protein (YAP), and kidney and brain expressed protein (KIBRA), three multivalent proteins with roles in protein scaffolding, cell polarity, memory performance, and cell proliferation. YAP and KIBRA each contain two tandem WW domains, a 40-residue interaction module composed of three antiparallel b-strands which recognize the Leucine/Proline-Proline-any amino acid-Tyrosine (L/PPXY) motif, three of which are present in a primarily disordered segment of AMOTL1. While several studies support co-localization of AMOTL1 with YAP and/or KIBRA in cultured cells, the structural basis of assembly in the context of all binding-competent domains is not well understood. Here, we show by multiple approaches including circular dichroism (CD), isothermal titration calorimetry (ITC), analytical size exclusion chromatography (SEC), and solution NMR that YAP and KIBRA bind AMOTL1 with micromolar affinity and have mapped the binding interfaces to the N and C-terminal L/PPXY motifs, respectively. The two KIBRA WW domains synergize to enhance binding to the AMOTL1 motifs but only one WW domain was required for assembly of the YAP-AMOTL1 complex. Our findings provide novel, molecular-level insights that extend understanding of intrinsically disordered regions and multivalent binding modules in protein complexes.